Azobenzene-diamides as Photopharmacological Ligands for Insect Ryanodine Receptor

Article abstract of DOI:10.1021/acs.jafc.0c03272

Photoresponsive ligands are powerful tool compounds for studying receptor function with spatiotemporal resolution. However, to the best of our knowledge, such a ligand is not available for the ryanodine receptor (RyR). Herein, we present a photochromic ligand (PCL) for insect RyR by decorating chlorantraniliprole (CHL) with photoswitchable azobenzene (AB). We demonstrated that one potent ligand, named ABCHL13, shows light-induced reversible trans-cis isomerization and 3.5-fold insecticidal activity decrease toward oriental armyworm (Mythimna separata) after UV-light irradiation, that is, trans-ABCH13 has higher activity than the cis-ABCH13. ABCHL13 enables optical control over intracellular Ca2+ release in dorsal unpaired median (DUM) neurons of M. separata and American cockroach (Periplaneta americana) and cardiac function of P. americana. Our results provide a first photopharmacological toolkit that is applicable to light-dependent regulation of RyR and heart beating.

Full text of DOI:10.1021/acs.jafc.0c03272

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Article
Azobenzene-diamides as Photopharmacological Ligands for Insect
Ryanodine Receptor
Long Wang, Meijun Chen, Shanshan Xia, Zhiping Xu, Yuxin Li,* and Xusheng Shao*
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ABSTRACT: Photoresponsive ligands are powerful tool compounds for studying receptor function with spatiotemporal resolution.
However, to the best of our knowledge, such a ligand is not available for the ryanodine receptor (RyR). Herein, we present a
photochromic ligand (PCL) for insect RyR by decorating chlorantraniliprole (CHL) with photoswitchable azobenzene (AB). We
demonstrated that one potent ligand, named ABCHL13, shows light-induced reversible trans−cis isomerization and 3.5-fold
insecticidal activity decrease toward oriental armyworm (Mythimna separata) after UV-light irradiation, that is, trans-ABCH13 has
higher activity than the cis-ABCH13. ABCHL13 enables optical control over intracellular Ca2+ release in dorsal unpaired median
(DUM) neurons of M. separata and American cockroach (Periplaneta americana) and cardiac function of P. americana. Our results
provide a first photopharmacological toolkit that is applicable to light-dependent regulation of RyR and heart beating.
KEYWORDS: photopharmacology, ryanodine receptor, azobenzene, chlorantraniliprole, insect
spatiotemporal resolution that other technologies cannot
confer. Various photopharmacological ligands have been
developed toward biologically important targets, opening a
new door for the research community to stimulate or silence
the function of target proteins.17 In calcium channel photo-
pharmacology, a couple of photochromic ligands (PCLs) were
developed, allowing for optical control over cardiac function
exemplified by photoswitchable diltiazem18 and muscarinic
agonist.19 However, the implementation of photopharmacol-
ogy in the pesticide arena is neglected. We recently developed
a series of photoresponsive pesticides, such as photocaged
fipronil20 and spirotetramat-enol,21 and photoswitchable
azobenzene-modified imidacloprid and benzoylphenylurea
analogues.22,23 Owing to the lack of photoresponsive ligands
for RyR, we report herein the first example of photochromic
insect RyR ligand by blending azobenzene (AB) with
chlorantraniliprole (CHL) and demonstrate its ability in
optical control over neuron and cardiac function of living
cockroach.
INTRODUCTION
■
Ryanodine receptors (RyRs) are intracellular homotetrameric
nonvoltage-gated calcium channels that are located in the
sarcoplasmic or endoplasmic reticulum (SR or ER) of both
vertebrates and invertebrates.1−4 RyRs play an essential role in
mediating the release of Ca2+ in many cell types, such as
neurons, epithelial cells, and muscle cells. Defective function of
RyRs is associated with heart failure, muscular dystrophy,
malignant hyperthermia, central core disease, and neuro-
degenerative disease.1−4 Currently, several mammalian and
invertebrate RyR crystal structures have been elucidated.5−7
RyRs respond to an array of antagonists (e.g., ryanodine,
dantrolene, and ruthenium red)8 and activators (e.g., p-
chlorocresol, suramin, caffeine, ATP, and cyclic ADP ribose).9
These ligands are basic tools for deciphering their role in
gating and regulation.
Insect RyRs are good models for physiological function
exploration and have attracted intensive attention recently.
Previous studies on RyRs from fruit flies, houseflies,
cockroaches, and diamondback moths indicate that insect
RyRs show a high degree of homology in amino acid sequence
with mammalian receptors.7,10 RyRs have three mammalian
isoforms (RyR1 to RyR3), while in insects, RyRs only have a
single isoform showing 47% identity at the amino acid level
with RyR2.7 Insect RyRs are the targets of a class of newly
developed insecticides called diamides whose global annual
sales approach US$2 billion.11,12 Diamide can activate the
receptor to induce the excessive release of Ca2+ from
intracellular stores located in SR, leading to insect feeding
cessation, lethargy, paralysis, and eventual death.13
MATERIALS AND METHOD
■
Synthetic Routes and Characterization. The chemicals and
1
instruments used, the detailed synthetic procedures, and H NMR,
13C NMR, and HRMS data are provided in the Supporting
Information.
Optical Properties. The target compound in acetonitrile (20
μM) in a quartz cuvette (1 cm × 1 cm) was irradiated with ultraviolet
Received: May 24, 2020
Revised: September 11, 2020
Accepted: November 13, 2020
Published: November 30, 2020
Photopharmacology is a fast-growing photonics-based
technology that relies on photoresponsive ligands to
manipulate biological functions.14−16 This technology provides
control over bioactivity or biological events with a
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© 2020 American Chemical Society
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(365 nm) at room temperature. The absorbance changes at different
time intervals were recorded with a UV−vis spectrophotometer until
they no longer changed. The ratio of cis/trans-isomers was measured
by ultraperformance liquid chromatography. The fatigue resistance
was determined by monitoring the absorbance change at the
maximum absorption through alternative irradiation with UV (λ =
365 nm) and blue light (λ = 430 nm). For the half-life test, the target
compound in acetonitrile (20 μM) was irradiated with UV to reach a
photostable state. The solution is stored in the dark and the
absorbance change at the maximum absorption wavelength was
measured by a UV−vis spectrophotometer at different time intervals.
All the operations have six repetitions.
Insecticidal Activity against M. separate and Aedes
albopictus Larvae. Insecticidal Test Method for Aedes albopictus
Larvae. The fourth-instar larvae of A. albopictus were provided by
Shanghai Southern Pesticide Creation Center. Two groups of aqueous
solutions with the same concentration gradient were prepared: one
was irradiated with UV (λ = 365 nm) for 30 min and the other group
was stored in the dark. Ten Aedes larvae of the same size were
selected and placed in a centrifugation tube. Then, the solution
containing different concentrations of chemicals was added to the
centrifugation tube, and the tube was placed in the dark in a
conditioned room (25 1 °C, 50% relative humidity (RH)). After 24
h treatment, the mortality of A. albopictus larvae was counted; the
larvae were considered to be dead unless they moved when touched
with a syringe. Water was used as a control. Each treatment had three
repetitions, and the data were adjusted and subjected to probit
analysis as before.
dissolved in dimethyl sulfoxide (DMSO) to give a certain
concentration of mother solution. The prepared solutions were
divided into two groups: one of which was irradiated with ultraviolet
(365 nm) for 30 min and the other group was stored in the dark. Ten
microliters of each solution was added to 1 mL of DUM neuron cell-
culture medium. After 6 h of incubation, 5 μM fluorescent dye
DiBAC4(3) was added for further incubation for 30 min, then the dye
was washed away. A 24-well plate containing DUM neuron cells was
immobilized on a laser confocal microscope stage to detect changes in
the fluorescence intensity of DUM neurons before and after
illumination. The excitation wavelength was 488 nm and the emission
wavelength was 525 nm, and each experiment was repeated three
times.
Intracellular Ca2+ Analysis in the Absence of Extracellular Ca2+.
PaDUM neurons in physiological solution C (1 mL) were placed in
four glass-bottom cell-culture dishes (35 mm). To the above four
dishes, 1 μL of DMSO, 1 μL of trans-ABCHL13 in DMSO without
irradiation, 1 μL of trans-ABCHL13 plus ruthenium red (10 μM), and
1 μL of ABCHL13 irradiated by UV light were added, respectively,
and then incubated for 60 min at 28 °C. The incubated neurons were
washed with phosphate-buffered saline (PBS), added with Fluo 3-AM
(10 μM), and incubated for 30 min at 28 °C. After incubation, the
neurons were photographed by a confocal laser scanning microscope
(Nikon Inc., Melville, NY). Emission was collected at 525 nm upon
excitation at 488 nm to observe changes in the fluorescence intensity
in DUM neuron cells. Three separate experiments were performed,
each with triplicate samples.
Intracellular Ca2+ Analysis in Mythimna separata. Isolation of
M. separata Neurons. M. separata Walker were initially obtained
from shallot fields in Tianjin, China, and reared indoors in climatic
chambers on an agar-based semisynthetic diet at 27 1 °C, 75 5%
relative humidity, and 16:8 h LD photocycle. The insects were reared
for two generations prior to the experiment. Third instar larvae of M.
separata were first anesthetized with 70% ethanol and their thoracic
and abdomen ganglia were removed and placed in saline. The thoracic
and abdomen ganglia were transferred to a solution containing 0.3%
trypsin for 6 min at 28 °C, plated into a 35 mm culture dish
containing 1 mL of improved L-15 Leibovitz culture medium
supplemented with fetal calf serum (15%, v−v), and then
mechanically dissociated using a fire-polished Pasteur pipette. The
cultures were maintained at 28 °C for 2 h to allow the cell to adhere
to the dish. All procedures were carried out under sterile conditions.
Intracellular Ca2+ Analysis. Calibration of the fluorescence signal
was achieved using the method reported by Takahashi et al. with
modifications. Briefly, the attached neurons were rinsed twice in
standard physiological saline [(mM): NaCl 150, KCl 4, MgCl2 2,
CaCl2 2, HEPES 10], buffered to pH 7.0 and then incubated in the
dark for 30 min at 28 °C in standard external saline containing the dye
Fluo 3-AM (10 μM) or incubated for 2 h with Fluo 5-N AM (10
μM). After dye loading, the cells were again rinsed in physiological
saline twice. Calcium-free extracellular fluid has the following
composition (mM), NaCl 150, KCl 4, MgCl2 2, EGTA 2, HEPES
10, buffered to pH 7.0. The new compound was applied after 3 min of
fluorescence recording. The original compound is eluted with
calcium-free extracellular fluid before the next compound is applied.
Calcium ratio imaging studies were conducted using the imaging
system coupled to an inverted fluorescence microscope with a Fluor
40 × oil immersion objective (Olympus IX71). The cells were excited
at 488 nm and the 530 nm fluorescence emission acquired using a
CCD (Image Pro-6.0). Each experiment was repeated at least six
times. The data were analyzed using GraphPad Prism on 7.0. The
Insecticidal Test for M. separate. The M. separate larvae were
reared in our laboratory, and the insecticidal activity was tested by the
leaf-dipping method. Two groups of solutions with the same
concentration gradient were prepared: one was irradiated with UV
(λ = 365 nm) for 30 min and the other group was stored in the dark.
The corn (Zea mays) leaves were well-immersed in the above-
prepared solutions, dried naturally in the dark, then placed in clean
culture dishes. Ten third instar M. separate larvae that were starved for
2 h were introduced into each dish. Then, the dishes were placed in
the dark for 72 h in a conditioned room (25 1 °C, 50% RH). The
mortality of the larvae was counted. The larvae were considered to be
dead unless they moved when touched with a syringe. Water was used
as a control. Each treatment had three repetitions, and the data were
adjusted and subjected to probit analysis as before.
Periplaneta americana Dorsal Unpaired Median (DUM)
Neuron Preparation. Physiological Solutions. A mixture of
physiological solution A, 185 mM NaCl, 3.0 mM KCl, 4 mM
MgCl2, 10 mM D-glucose, 10 mM N-(2-hydroxyethyl)piperazine-N′-
ethanesulfonic acid (HEPES) was adjusted to pH 7.2 with NaOH and
stored at 4 °C. Physiological solution B was prepared by adding type
IA collagenase 1.5 mg mL−1 to solution A. Physiological solution C
was prepared by adding 5 mM CaCl2, fetal calf serum (10% by
volume), and double antibody [1% penicillin (50 IU mL−1)/
streptomycin (50 mg mL−1)] to solution A.
Preparation of DUM Neurons of P. americana. The body surface
of male P. americana adults (purchased from Tengfei Cockroach
Company, Anhui, China) was disinfected with 75% alcohol. Under a
dissecting microscope, the body wall was cut along the midline and
fixed in a wax plate. The impurities such as trachea, digestive tract,
and fat body were carefully removed and the ganglion was dissected in
physiological solution A. The whole process is operated on a clean
bench. The obtained ganglion was transferred to physiological
solution B and hydrolyzed at 37 °C for 20 min. After the enzymatic
hydrolysis was completed, the ganglion was transferred to
physiological solution C, and the digestion was terminated by rinsing
three times with physiological solution C. The ganglion was
repeatedly blown into physiological solution C to disperse the cells
in the ganglion. The obtained cell suspension was filtered through a
200-mesh filter into a Petri dish containing physiological solution C at
28 °C.
results were expressed as mean
SD (n = number of cells).
Fluorescence values were expressed as F/F0, F0 being the resting (or
baseline) fluorescence and F the change in fluorescence from baseline
after the drug application.
Photomodulation of P. americana Heartbeat. Four male P.
americana were fixed by two pins with back up and their wings and
legs were cut off. Then, their first and second terga were sequentially
lifted up and cut off, and the fat was carefully removed and washed off
by physiological saline (pH = 7.2). The separated terga with the heart
Intracellular Ca2+ Analysis in PaDUM Neurons. Intracellular
Ca2+ Analysis in the Presence of Extracellular Ca2+. ABCHL13 was
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Figure 1. RyR PCLs are derived from RyR activator chlorantraniliprole and photoswitch azobenzene. Ligands ABCHL1−9 and ABCHL11 are
designed by the replacement of amide bond with azobenzene. ABCHL10 is a divalent compound linked by azobenzene. ABCHL12−13 are
generated by the exterior attachment of azobenzene.
was transferred to a cell-culture dish containing physiological solution
(NaCl 200 μM, KCl 10.7 μM, MgSO4 0.1 μM, NaHCO3 2.1 μM,
NaH2PO4 0.08 μM, and HEPES 10 g·L−1) (3 mL) and incubated for
30 min at 25 °C. Then, the separated terga were treated with DMSO
(3 μL), trans-ABCHL13 (100 μM), trans-ABCHL13 (100 μM), and
UV irradiation (10 min) and chlorantraniliprole (100 μM),
respectively, incubated for 10 min at 25 °C. Then, the heartbeat
rates in 1 min were calculated after 5, 10, 20, and 30 min treatment.
Each experiment was repeated six times.
Photophysiochemical Properties. With the target
compounds in hand, we set out to investigate their photo-
physiochemical properties using UV−vis spectroscopy and
high-performance liquid chromatography (HPLC) analysis to
verify whether they are suitable for further biological tests. The
half-lives of azobenzene analogues were calculated based on
the change in absorbance at 336 nm (λtrans‑max π−π*) over time.
The isosbestic points of the trans- and cis-structure were
determined by UV−vis spectroscopy, which are taken as the
detection wavelength of HPLC to calculate the percentages of
RESULTS AND DISCUSSION
■
both isomers. The maximum absorbance wavelength (λmax
,
Molecular Design and Synthesis. Diamides are a new
chemotype of RyR activator. The currently developed diamides
are divided into two categories: phthalic diamides exemplified
by flubendiamide13 and anthranilic diamides represented by
cyantraniliprole and CHL.24,25 CHL is now the largest
insecticide with sales volume over US$1 billion. CHL has a
high binding affinity with insect RyR.26 We therefore used
CHL as a prototype for structural modification by introducing
photoswitchable azobenzene at different positions to confer
photosensitivity. Bioisosteric replacement and azo-extension
strategy were employed in our molecular modification. CHL
contains three structural segments: benzene ring, two amides,
and pyridine−pyrazole. Previous investigations proved that aryl
benzamide is a bioisosteric group of azobenzene that has been
successfully applied for putting bioactive molecules under
optical control.27,28 We first replaced two amide groups in
CHL with azobenzene-generating compounds ABCHL1−9
and ABCHL10, respectively. We then linked two CHL
molecules together by diazene unit giving a divalent compound
ABCHL11. Attaching azobenzene at the exterior of bioactive
molecule is also an effective strategy.29 Therefore, we prepared
analogues ABCHL12 and ABCHL13 by introducing azoben-
zene at the phenyl ring of CHL (Figure 1).
nm), the ratio of photoisomers, and the thermal relaxation half-
lives are summarized in Table 1. All of the compounds show
typical azobenzene absorption band and photoswitching
behaviors with the maximum absorption wavelength at around
365 nm. ABCHL1−9 showed poor photoisomerization
efficiency (39−54% conversion from trans to cis configuration)
and their cis-isomers were not stable enough with 1−2 h
thermal relaxation half-lives. The thermal instability of a cis-
isomer was probably caused by substituent effects of ortho-
amide as the substituents at the ortho-position would attenuate
cis-isomer’s stability.30,31 ABCHL10 and ABCHL11 have
improved isomerization efficiency but their cis forms still
have low thermal stability. ABCHL12 and ABCHL13 can
gradually transform to their cis forms (>90% conversion rates)
upon UV irradiation (Figure 3A) and thermal back half-lives
were 26 and 11 h, respectively, indicating suitable photo-
switching properties for further biological studies. ABCHL13 is
resistant to photobleaching and could be switched back and
forth more than 15 times (Figure 3B).
Insecticidal Activity. To identify the performance of our
RyR PCLs, we evaluated their insecticidal activities using
armyworm (M. separate) and mosquito larvae (A. albopictus).
We initially screened the insecticidal activity of trans-isomers.
Toward M. separate, trans-ABCHL1−12 almost lost insectici-
dal activity (Table 2). Fortunately, ABCHL13 showed 100%
mortality at 500 mg L−1. These results demonstrate that the
replacement of aryl amide with azobenzene or preparing
divalent ligand is not applicable for maintaining activity. As cis-
ABCHL1−9 have low thermal stability,it is difficult to test
their accurate activities. We therefore selected ABCHL10−13
to further investigate the insecticidal activities of both
photoisomers. No obvious insecticidal activities were observed
The synthetic routes of target compounds are depicted in
Figure 2. Compounds ABCHL1−9 were prepared from o-
diaminobenzene. ABCHL10 was prepared from intermediates
6 and 9. Divalent ABCHL11 and monovalent ABCHL12 were
synthesized by four-step and five-step procedures, respectively.
Finally, ABCHL13 was obtained through a seven-step
procedure using 2-amino-3-methylbenzoic acid as the starting
1
material. All of the compounds were well-characterized by H
NMR, 13C NMR, and HRMS.
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Figure 2. Synthesis routes of ABCHLs.
for ABCHL10−12 before and after irradiation. Both isomers of
ABCHL13 have almost the same activity against mosquito
larvae, while they exhibited 3.5-fold activity difference toward
M. separate (Figure 4A), indicating that ABCHL13 can be
photoinactivated upon light irradiation. In control experiments,
light has no perturbance on the activity of the precursor CHL.
Recently, bee toxicity has become a big concern. Therefore, we
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Table 1. Maximum Absorbance Wavelength (λmax, nm), the
Ratio of Trans- and Cis-Isomers and the Thermal
Relaxation Half-Lives of ABCHLs
Table 2. Insecticidal Activity of ABCHLs against A.
albopictus and M. separate
insecticidal activity (mortality at 500 mg L−1
)
compound
nonirradiated trans:cis
irradiated trans:cis
t1/2 (h)
compound
A. albopictus
M. separate
ABCHL1
ABCHL2
ABCHL3
ABCHL4
ABCHL5
ABCHL6
ABCHL7
ABCHL8
ABCHL9
ABCHL10
ABCHL11
ABCHL12
ABCHL13
93: 7
81:19
81:19
80:20
82:18
95:5
88:12
80:20
93:7
46: 54
50:50
46: 54
54:46
60:40
63:37
56:44
55:45
61:39
38:62
21:79
5:95
1.5
1.4
1.0
2.0
1.8
1.6
1.8
1.3
1.6
3.6
56
ABCHL1
ABCHL2
ABCHL3
ABCHL4
ABCHL5
ABCHL6
ABCHL7
ABCHL8
ABCHL9
ABCHL10
ABCHL11
ABCHL12
ABCHL13
CHL
100
100
100
80
100
90
100
100
20
0
0
0
100
100
0
80
50
80
70
60
80
70
80
30
20
0
1
3
4
2
1
2
1
1
2
5
2
3
2
100:0
100:0
97:3
26
11
0
100
100
100:0
9:91
azobenzene
evaluated bee toxicity of ABCHL13 using BeeTox,32,33 and the
results showed that it is not toxic (87.22% negative) to bees.
ABCHL13 Enables Optical Modulation of Neurons.
Having identified the in vivo activity difference, we sought to
investigate its ability for optically modulating insect RyR using
M. separate neurons and American cockroach (P. americana)
DUM neurons. We used fluorescence dye Fluo 3-AM as an
indicator for monitoring ratiometric Ca2+ inside the cells. Fluo
3-AM is an intracellular Ca2+ fluorescent probe that can enter
the cytoplasm and be enzymatically hydrolyzed into Fluo 3.
The free Fluo 3 has a weak fluorescence. However, when Fluo
3 chelates Ca2+, its fluorescence intensity enhances the
proportionally to Ca2+ concentration. Diamide insecticides
can keep the ryanodine receptor open, resulting in a sharp
increase in Ca2+ in the cytoplasm. When M. separate neurons
were loaded with Fluo 3-AM and treated with trans-ABCHL13
(5 μM), the peak value of F/F0 (%) was 102.0 0.41 (n =
12). After irradiation, the peak value of F/F0 (%) decreased to
99.8 0.15 (n = 12) (Figure 4B), which is almost equal to that
of the control. From this observation, we concluded that trans-
ABCHL13 can be photoinactivated to induce the opening of
the calcium channel in an on−off manner. The same trend was
also observed for neurons treated with 20 μM ABCHL13
(Figure 4C). After the neurons of M. separate were incubated
with ryanodine for 5 min and then treated with trans-
ABCHL13 (20 μM) and light, the peak value of F/F0 (%)
decreased from 106.1 1.61 (n = 12) to 104.63 1.87 (n =
12, Figure 4D), indicating that ryanodine can perturb the
binding affinity of our ligand. These experiments implied that
RyRs in the neuron ER are the possible targets of trans-
ABCHL13 (Table 3).
Next, we used DiBAC4(3) to monitor the intracellular cation
changes in PaDUM neurons. When PaDUM neurons were
subjected to trans-ABCHL13 (10 μM) and the dye, strong
fluorescence was observed (fluorescence intensity, 49.6). Upon
irradiation, 1.5-fold fluorescence intensity reduction (32.9) was
detected (Figure 5A). To further identify our ligand acts on
insect RyRs, we used ruthenium red (RuR) as the RyR
inhibitor8 and Fluo 3-AM as a probe to measure the Ca2+-
releasing ability in acute separation P. americana DUM
neurons. RuR is a noncompetitive inhibitor of the ryanodine
receptor. In the extracellular absence of Ca2+, the PaDUM
neurons treated with RuR and Fluo 3-AM do not emit
fluorescence. After adding trans-ABCHL13 to the above-
treated neurons, the fluorescence gradually resumed with an
intensity of 16.9 (Figure 5B), indirectly indicating that our
ligand acts on insect RyR. While the corresponding
fluorescence intensity recovered by cis-ABCHL13 was 5.46,
suggesting a 3.42-fold decrease in activity toward RyR. The
above results implied that ABCHL13 alone can open the
channel and cause an increase in the intracellular Ca2+
concentration.
ABCHL13 Enables Optical Modulation of Heart
Pacing. After establishing the light-dependent inactivation of
Figure 3. UV−vis spectroscopy analysis. (A) UV−vis absorption spectra change of ABCHL13 (20 μM) upon UV (356 nm) irradiation. (B)
Fatigue resistance of ABCHL13 (20 μM) by alternative irradiation of UV light and (purple lines) and blue light (blue lines).
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Figure 4. Insecticidal activity and Ca2+-releasing ability of ABCHL13. (A) Insecticidal activity ABCHL13 toward A. albopictus and M. separate
before and after irradiation. (B−D) Ca2+-releasing ability of ABCHL13 in M. separate neurons using fluorescence dye Fluo 3-AM as an indicator at
5 μM (B) and 20 μM (C) and in the presence of ryanodine (D).
Table 3. Insecticide Activity of ABCHLs against A. albopictus and M. separate before and after UV Irradiation
A. albopictus
M. separate
compound
ABCHL10
LC50 (mg L−1
)
y = ax + b
LC50 (mg L−1
)
y = ax + b
a
light
dark
light
dark
light
dark
light
dark
light
dark
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
73.09
21.13
0.21
0.21
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
23.71
24.58
ABCHL11
ABCHL12
ABCHL13
CHL
y = 2.6252x + 1.7275
y = 2.6884x + 1.6627
y = 3.4436x + 0.8348
y = 4.0840x + 0.6914
y = 5.7001x + 1.0385
y = 5.7801x + 1.1408
CK
n.a.
a
n.a. = no activity.
insect RyR in neurons, we turned to investigate its ability to
optically control cardiac functions. Diamides regulate the
release of stored intracellular calcium and play a critical role in
muscle contraction and insect heart beating. P. americana has
an open circulatory system with a heart (an elongated
contractile tube) lying along the mid-dorsal line beneath the
terga. The heart contraction was controlled by alary muscles.
The back of P. americana containing the heart was cut off and
stored in physiological solution followed by quick addition of
ABCHL13. After 5 min of incubation, the heartbeat rate was
calculated every 5 min (Figure 5C). After the application of
trans-ABCHL13, the heartbeat rate reduced about 71% (n = 5
hearts) in comparison with untreated control. Upon
illumination with UV light, the heartbeat rate reduced to
65% (Figure 5D), that is, the cis-isomer has a lower ability to
reduce heartbeat rate than the trans-isomer. After 30 min of
treatment, the difference in the heart pacing between trans-
and cis-ABCHL13 was 1.13-fold, which is consistent with the
difference observed in calcium-releasing ability. Both isomers
had a higher influence on the heartbeat rate than CHL,
although the in vivo insecticidal activity of ABCHL13 was
lower than that of CHL. The above observations demonstrated
that ABCHL13 enables remote and optical control of heart
rate in the cockroach.
Cardiac pacing plays an important role in the circulatory
system, and its malfunction relates to many serious diseases.
Cardiac rhythm control drug is the most commonly used
strategy to steady the heartbeat and ease symptoms. Cardiac
function is controlled by the nervous system and cardiac
muscle. For optical control of cardiac function, photo-
switchable diltiazem18 and muscarinic agonist19 were devel-
oped toward L-type Ca2+ channels and muscarinic acetylcho-
line receptors, respectively. Our results established here for the
first time that RyR is an alternative target for optical control of
cardiac function. As RyRs exist both in vertebrates and
invertebrates, the information gained from invertebrates may
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J. Agric. Food Chem. 2020, 68, 14409−14416

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Article
Figure 5. (A) Fluorescence images of PaDUM neuron treated with ABCHL13 and Fluo 3-AM. (B) Fluorescence images of PaDUM neuron
treated with RuR, ABCHL13, and Fluo 3-AM. (C) P. americana heart of the control and insect treated with CHL, ABCHL13 alone, and ABCHL13
plus light. (D) Heartbeat rate of P. americana treat with CHL and ABCHL13 before and after irradiation.
provide guidelines for developing new therapeutic agents in
vertebrates.
University, Tianjin 300071, China; Email: liyx128@
nankai.edu.cn
In conclusion, we present a first example of the photo-
chromic RyR ligand via coupling of azobenzene with diamide
CHL. ABCHL13 can be photoinactivated upon irradiation and
showed differential insecticidal activity toward M. separate and
different Ca2+-releasing ability in M. separate and PaDUM
neurons by acting on insect RyR. Furthermore, ABCHL13 can
be used for real-time modulation of the heartbeat of cockroach.
Although the difference in the activity was not high enough,
our results implied that RyR PCLs are applicable for cardiac
function control. We hope the information we gained here
would provide guidelines for the future development of PCLs
toward mammalian RyR to enable spatiotemporal control over
cardiac function.
Authors
Long Wang − Shanghai Key Laboratory of Chemical Biology,
School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
Meijun Chen − Shanghai Key Laboratory of Chemical
Biology, School of Pharmacy, East China University of
Science and Technology, Shanghai 200237, China
Shanshan Xia − Shanghai Key Laboratory of Chemical
Biology, School of Pharmacy, East China University of
Science and Technology, Shanghai 200237, China
Zhiping Xu − Shanghai Key Laboratory of Chemical Biology,
School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c03272.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c03272
■
sı
Funding
This work was financially supported by the National Key
Research and Development Program of China
(2018YFD0200100), the National Natural Science Foundation
of China (No. 21877039), and the Innovation Program of
Shanghai Municipal Education Commission (2017-01-07-00-
02-E00037).
1
Synthetic procedures and H NMR, 13C NMR, and
HRMS of new compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Xusheng Shao − Shanghai Key Laboratory of Chemical
Biology, School of Pharmacy and State Key Laboratory of
Bioreactor Engineering, East China University of Science and
Technology, Shanghai 200237, China; orcid.org/0000-
0002-2579-7533; Phone: +86-21-64253540;
Email: shaoxusheng@ecust.edu.cn; Fax: +86-21-64252603
Yuxin Li − State Key Laboratory of Elemento-Organic
Chemistry, Institute of Elemento-Organic Chemistry, Nankai
Notes
The authors declare no competing financial interest.
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