Cell active and functionally-relevant small-molecule agonists of calcitonin receptor

Article abstract of DOI:10.1016/j.bioorg.2020.103596

The natural calcitonin (CT) receptor and its peptide agonists are considered validated targets for drug discovery. A small molecule agonist, SUN-B8155, has previously been shown to efficiently activate cellular CTR. Herein, we report the synthesis of a se

Full text of DOI:10.1016/j.bioorg.2020.103596

BioorganicChemistry96(2020)103596
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Cell active and functionally-relevant small-molecule agonists of calcitonin
receptor
Shuai Zhaoa,1, Shengchao Guob,1, Chan Yangc,1, Zheng Gongb,1, Yaomin Wanga, Yingli Jiac,
⁎
⁎
⁎
Xinyu Jianga, Liwei Xug, Li Shig, Xiao Yud,f, Jinpeng Sunb,c, , Yan Zhange, , Xin Chena,
a Department of Medicinal Chemistry, School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu 213164, China
b Key Laboratory Experimental Teratology of the Ministry of Education and Department of Biochemistry and Molecular Biology, Shandong University School of Medicine,
Jinan, Shandong 250012, China
c Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Key Laboratory of Molecular Cardiovascular Science, Ministry of
Education, Beijing 100191, China
d Key Laboratory Experimental Teratology of the Ministry of Education and Department of Physiology, Shandong University School of Medicine, Shandong 250012, China
e Department of Pathology of Sir Run Run Shaw Hospital and Department of Biophysics, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China
f Department of Physiology, Shandong University School of Medicine, Jinan, Shandong 250012, China
g Department of Tumor Hematology, The First Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, Jilin 130021, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
CTR
The natural calcitonin (CT) receptor and its peptide agonists are considered validated targets for drug discovery.
A small molecule agonist, SUN-B8155, has previously been shown to efficiently activate cellular CTR. Herein, we
report the synthesis of a series of compounds (S8155 1-9) derived from SUN-B8155, and investigate the struc-
tural-functional relationship, bias properties and their cellular activity profile. We discover that the N-hydroxyl
group from the pyridone ring is required for G protein activity and its affinity to the CT receptor. Among the
compounds studied, S8155-7 exhibits improved G protein activity while S8155-4 displays a significant β-ar-
restin-2 signaling bias. Finally, we show that both S8155-4 and S8155-7 inhibit tumour cell invasion through
CTR activation. These two compounds are anticipated to find extensive applications in chemical biology research
as well drug development efforts targeting CT receptor.
Agonist
GPCR
Bias
Migration
1. Introduction
calcitonin peptides, including KBP-042, KBP-088 and KBP-089, which
not only preserved the high efficacy of salmon calcitonin but also
CTR is the most ancient member of the class B GPCR family and is
widely expressed in many tissues of the human body [1–3]. It is known
that the CTR has multiple functions, including but not limited to
maintaining the quiescent state of muscle stem cells [4], limiting bone
loss, promoting osteoclast survival [5–7], regulating brain function, and
contributing to cancer progression [8–14]. Therefore, it is not sur-
prising that CTR has been an important therapeutic target for the
treatment of osteoporosis, Paget’s disease, hypercalcaemia of malig-
nancy, and cancer [3,15].
showed increased tolerability in rats [15–19]. However, due to the cost
and to potential side effects with unclear mechanisms, these peptide-
based therapies still experience limited use [16]. Alternatively, small-
molecule agonists of CTR with defined signalling pathways, which may
have a longer half-life in the body than peptide agonists, have broader
application potentials. To date, only one small-molecule agonist SUN-
B8155 was reported to effectively activate CTR [20]. CTR activates
both G proteins and β-arrestin-2 signalling [21–36]. It was proposed
that the functions of inhibition of cancer progression and limiting bone
loss mediated by CTR are dependent on Gs signalling [11,10,15].
However, functional selectivity of different downstream effectors, such
as arrestins downstream of CTR have not been elucidated yet, partially
The natural CTR peptide agonists derived from both human and
salmon calcitonin have been used clinically to treat osteoporosis and
Paget’s disease [15,16]. Recent studies have also developed mimetic
⁎ Corresponding authors at: Key Laboratory Experimental Teratology of the Ministry of Education and Department of Biochemistry and Molecular Biology,
Shandong University School of Medicine, Jinan, Shandong 250012, China (J. Sun).
E-mail addresses: sunjinpeng@sdu.edu.cn (J. Sun), zhang_yan@zju.edu.cn (Y. Zhang), xinchen@cczu.edu.cn (X. Chen).
URLs: http://www.yuxiao-sunjinpeng-lab.org/Home/ (J. Sun), https://person.zju.edu.cn/zhangyan (Y. Zhang),
http://life.cczu.edu.cn/2017/0515/c11922a157049/page.htm (X. Chen).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.103596
Received 3 October 2019; Received in revised form 18 January 2020; Accepted 19 January 2020
Availableonline21January2020
0045-2068/©2020PublishedbyElsevierInc.

S. Zhao, et al.
BioorganicChemistry96(2020)103596
due to the lack of functionally-selective CTR agonists.
(s, 1H), 6.57 (d, J = 6.0 Hz, 1H), 6.43 (s, 2H), 5.68 (s, 1H), 5.43 (s, 2H),
2.48 (s, 3H), 2.37 (s, 3H). 13C NMR (75 MHz, DMSO‑d6): δ 168.8, 164.3,
158.9, 150.0, 148.6, 137.3, 129.9, 113.0, 112.6, 110.1, 109.8, 98.9,
25.2, 20.7. HRMS (ESI, positive) Calcd for C14H15N3O3H [M+H]+
274.1186, found: 274.1184.
Recently, development of biased agonists with functional se-
lectivity, which signal with different efficacies to a particular receptor’s
multiple downstream pathways (e.g. individual G protein subtype or β-
arrestin pathways) are useful tools for dissection of specific signaling
pathways[29,30,49–52]. These biased ligands may also possess better
therapeutic potential because they can avoid “harmful” effect selec-
tively, compared to “balanced agonists” [46,47]. Therefore, develop-
ment of small molecules eliciting biased CTR functions will be valuable
tools in studying selective CTR downstream functions. However, the β-
arrestin activity of the only known CTR small-molecule agonist SUN-
B8155 has not been fully characterized [20].
S8155-4 (R = 4-NH2): 63% yield, pale yellow solid. Mp 251–253
°C. 1H NMR (300 MHz, DMSO‑d6): δ 14.62 (s, 1H), 10.04 (s, 1H), 6.95
(d, J = 8.7 Hz, 2H), 6.63 (d, J = 8.7 Hz, 2H), 5.63 (s, 1H), 5.42 (s, 2H),
2.43 (s, 3H), 2.34 (s, 3H). 13C NMR (75 MHz, DMSO‑d6): δ 168.9, 164.0,
158.8, 148.5, 148.4, 126.3, 124.5, 114.0, 109.3, 98.7, 25.2, 20.6;
HRMS (ESI, positive) Calcd for C14H15N3NaO3 [M+Na]+ 296.1006,
found: 296.1004.
In the present study, we synthesized a series of compounds (S8155-1
to S8155-9) based on the chemical structure of SUN-B8155, and in-
vestigated the bias property using SUN-B8155 as control. Compound
S8155-7 displayed a Gs biased property and at least five SUN-B8155
derivatives exhibited β-arrestin-2 biased signalling properties. Cellular
transwell studies demonstrated that compounds SUN-B8155, S8155-4
and S8155-7 inhibited breast cancer cells (MCF-7) migration via acti-
vation of CTR.
S8155-5 (R = 2-MeO): 76% yield, pale yellow solid. Mp 238–240
°C. 1H NMR (300 MHz, DMSO‑d6): δ 14.72 (s, 1H), 10.09 (s, 1H),
7.38–7.31 (m, 2H), 7.21–7.19 (m, 1H), 7.08–7.05 (m, 1H), 5.67 (d,
J = 0.6 Hz, 1H), 3.84 (s, 3H), 2.43 (s, 3H), 2.37 (s, 3H). 13C NMR
(75 MHz, DMSO‑d6): δ 169.1, 164.4, 158.9, 153.0, 148.4, 128.9, 127.0,
125.2, 120.6, 112.3, 110.0, 99.3, 55.9, 25.3, 20.7. HRMS (ESI, positive)
Calcd for C15H16N2NaO4 [M+Na]+ 311.1002, found: 311.1004.
S8155-6 (R = 3-Cl): 67% yield, pale yellow solid. Mp 204–206 °C.
1H NMR (300 MHz, CDCl3): δ 14.84 (s, 1H), 9.24 (s, 1H), 7.41–7.31 (m,
2H), 7.21 (s, 1H), 7.11 (d, J = 7.5 Hz, 1H), 5.81 (s, 1H), 2.46 (s, 3H),
2.37 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 168.8, 162.1, 158.1, 148.3,
137.9, 135.4, 130.8, 128.3, 126.2, 124.3, 110.3, 100.0, 26.2, 21.2.
HRMS (ESI, positive) Calcd for C14H13ClN2NaO3 [M+Na]+ 315.0507,
found: 315.0508.
2. Materials and methods
2.1. Chemical synthesis
2.1.1. Synthetic procedure for 5-acetyl-1,6-dihydroxy-4-methyl-2(1H)-
pyridone (1)
S8155-7 (R = 2-Me): 45% yield, pale yellow solid. Mp 197–199 °C.
1H NMR (300 MHz, CDCl3): δ 14.74 (s, 1H), 9.24 (s, 1H), 7.33–7.26 (m,
3H), 7.15–7.12 (m, 1H), 5.85 (s, 1H), 2.42 (s, 3H), 2.41 (s, 3H), 2.30 (s,
3H). 13C NMR (75 MHz, CDCl3): δ 169.7, 161.9, 158.0, 148.3, 135.7,
134.0, 131.4, 128.6, 127.0, 126.7, 109.4, 99.5, 26.2, 21.0, 18.1. HRMS
(ESI, positive) Calcd for C15H16N2NaO3 [M+Na]+ 295.1053, found:
295.1053.
Hydroxylamine hydrochloride (9 g, 130 mmol), water and acet-
onitrile (2:1 v/v, 6 mL) were added to a 250 mL three-necked flask. At 0
°C, triethylamine (60 mL, 260 mL) was slowly added to the reaction
mixture, and then diketene (20 mL, 260 mmol) dissolved in a mixture of
water and acetonitrile (2:1 v/v, 12 mL) was added dropwise to the
reaction solution while the temperature was maintained below 5 °C.
After the completion of addition of diketene solution, the reaction
mixture was stirred at 0 °C for 30 min, and then allowed to stand at
ambient temperature for 1 h. 6 N HCl aqueous solution was slowly
added to the reaction mixture to adjust the pH to 7 that resulted in
formation of a white suspension. The precipitated solid was filtered and
washed with water, and the crude product was recrystallized from
ethanol to obtain pale yellow solid 1 (11.9 g, 50% yield). Mp 195–197
°C. 1H NMR (300 MHz, DMSO‑d6): δ 5.81 (s, 1H), 2.56 (s, 3H), 2.32 (s,
3H). 13C NMR (75 MHz, DMSO‑d6): δ 194.3, 165.1, 157.9, 149.1, 107.6,
104.7, 28.0, 23.3.
S8155-8 (R = 3-Br): 50% yield, pale yellow solid. Mp 227–229 °C.
1H NMR (300 MHz, DMSO‑d6): δ 14.86 (s, 1H), 10.14 (s, 1H), 7.62–7.57
(m, 2H), 7.45 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 5.71 (s, 1H),
2.47 (s, 3H), 2.36 (s, 3H). 13C NMR (75 MHz, DMSO‑d6): δ 168.6, 164.4,
158.9, 148.5, 138.5, 131.3, 130.2, 128.5, 124.9, 122.0, 110.6, 99.7,
25.1, 20.9. HRMS (ESI, positive) Calcd for C14H13BrN2O3H [M+H]+
337.0182, found: 337.0180.
S8155-9: 71% yield, pale yellow solid. Mp 231–233 °C. 1H NMR
(300 MHz, DMSO‑d6): δ 15.24 (s, 1H), 10.23 (s, 1H), 8.08–7.90 (m, 3H),
7.66–7.58 (m, 4H), 5.75 (s, 1H), 2.42 (s, 3H), 2.39 (s, 3H). 13C NMR
(75 MHz, DMSO‑d6): δ 169.9, 164.8, 158.9, 148.6, 133.8, 132.8, 128.6,
128.4, 128.1, 127.7, 127.0, 125.7, 124.3, 121.9, 110.3, 99.6, 25.2,
2.1.2. General procedure for synthesizing SUN-B8155 and its derivatives
(S8155-1, -2, -4-9)
20.9. HRMS (ESI, positive) Calcd for
331.1053, found: 331.1049.
C
18H16N2NaO3 [M+Na]+
A mixture of 1 (300 mg, 1.6 mmol) and substituted aniline
(1.6 mmol) in EtOH (12 mL) was heated at 80 °C for 3 h. After the
reaction was completed, the reaction solution was cooled to ambient
temperature, resulting in precipitates. The solid was obtained by suc-
tion filtration, washed with cold ethanol and air-dried. SUN-B8155
(R = 2-NH2): 75% yield, pale yellow crystal. Mp 196–198 °C. 1H NMR
(300 MHz, DMSO‑d6): δ 14.36 (s, 1H), 10.06 (s, 1H), 7.12–7.07 (m, 1H),
7.00 (dd, J = 7.8 Hz, 1.5 Hz, 1H), 6.82 (dd, J = 8.1, 1.2 Hz, 1H),
6.65–6.60 (m, 1H), 5.65 (s, 1H), 5.26 (s, 2H), 2.37 (s, 6H). 13C NMR
(75 MHz, DMSO‑d6): δ 170.7, 164.3, 158.8, 148.6, 144.0, 128.8, 127.1,
121.4, 116.3, 115.8, 109.3, 99.2, 25.3, 20.2. HRMS (ESI, positive)
Calcd for C14H15N3NaO3 [M+Na]+ 296.1006, found: 296.1007.
S8155-1 (R = 4-F): 40% yield, pale yellow solid. Mp 215–216 °C.
1H NMR (300 MHz, DMSO‑d6): δ 14.72 (s, 1H), 10.05 (s, 1H), 7.34–7.23
(m, 4H), 5.61 (s, 1H), 2.35 (s, 3H), 2.28 (s, 3H). 13C NMR (75 MHz,
DMSO‑d6): δ 169.1, 164.4, 162.6, 159.3, 159.0, 148.6, 133.2, 128.0,
127.9, 116.6, 116.3, 99.3, 25.2, 20.7. HRMS (ESI, positive) Calcd for
2.1.3. Synthetic procedure for S8155-3
A
50 mL round-bottom flask was charged with 1 (300 mg,
1.6 mmol), Raney nickel (30 mg), and methanol (15 mL), and the re-
action was carried out at ambient temperature and under H2 atmo-
sphere for 12 h. After the reaction was completed, the catalyst was
removed by filtration, and the crude product was recrystallized to af-
ford 5-acetyl-6-hydroxy-4-methyl-2(1H)-pyridone (2) (207 mg, 80%
yield) as white crystal. 1H NMR (300 MHz, DMSO‑d6): δ 12.07 (s, 1H),
5.85 (s, 1H), 2.70 (s, 3H), 2.49 (s, 3H); 13C NMR (75 MHz, DMSO‑d6): δ
195.0, 169.3, 161.5, 151.9, 110.4, 103.8, 28.1, 23.5.
To a 50 mL round bottom flask was added 2 (196 mg, 1.3 mmol), O-
phenylenediamine (138 mg, 1.3 mmol) and ethanol (10 mL), and the
mixture was heated to reflux for 3 h. After the disappearance of the
starting material, the reaction solution was cooled to ambient tem-
perature, and the resulting solid (213 mg, 60% yield) was obtained by
suction filtration, washing and air-drying.
C
14H13FN2O3H [M+H]+ 277.0983, found: 277.0984.
S8155-2 (R = 3-NH2): 63% yield, pale yellow solid. Mp 214–216
S8155-3: Mp 230–232 °C. 1H NMR (300 MHz, DMSO‑d6): δ 14.56 (s,
1H), 10.75 (s, 1H), 7.10–7.04 (m, 1H), 6.98–6.95 (m, 1H), 6.82–6.80
°C. 1H NMR (300 MHz, DMSO‑d6): δ 14.77 (s, 1H), 10.10 (s, 1H), 7.12
2

S. Zhao, et al.
BioorganicChemistry96(2020)103596
(m, 1H), 6.64–6.58 (m, 1H), 5.52 (s, 1H), 5.23 (s, 2H), 2.35 (s, 3H),
2.33 (s, 3H). 13C NMR (75 MHz, DMSO‑d6): δ 169.8, 168.6, 163.1,
152.1, 143.9, 128.6, 127.2, 121.6, 116.2, 115.7, 110.9, 99.2, 25.4,
19.9. HRMS (ESI, positive) Calcd for C14H15N3O2H [M+H]+ 258.1237,
found: 258.1241.
instructions. After 24 h, transfected cells were added to the upper
chamber and cultured in 10% FBS, while the lower chamber was sup-
plied with DMEM containing 10% FBS and different CTR ligands. After
48 h, cells on the lower surface of membrane were fixed and stained
with crystal violet. For each well, cell counting was performed in ten
randomly selected fields with absorbance measured at 490 nm. Each
experiment was performed in triplicate and repeated at least three
times. sCT is the agonist of CTR and SCT 8-32 is the antagonist of CTR,
both of which were used at a concentration of 1 μM. SUN-B8155,
S81554 and S8155-7 were used at a concentration of 10 μM.
2.2. Constructs
Wild-type human CTR was generated by DNA synthesis and mod-
ified to include a C-terminal YFP tag in a mammalian expression vector
pcDNA3.1. Plasmid encoding β-arrestin-2 was a generous gift from Dr.
R.J Lefkowitz at Duke University. The GloSensor plasmid was obtained
from Promega. All constructs were verified by DNA sequencing.
2.7. Statistics
All data were present as mean
SEM. The EC50 values for cAMP
assays and BRET assays were calculated using Graphpad Prism 6 soft-
ware. The differences between groups in Transwell® assays were ana-
lyzed using one-way analysis of variance (one-way ANOVA). P-values
less than 0.05 were statistically significant.
2.3. Measurements of cAMP accumulation
HEK293 cells were co-transfected with CTR-YFP and GloSensor
using PEI (Polysciences) according to the manufacturer’s instructions.
Twenty-four hours later, transfected cells were seeded into 96-well
plates at a density of 30,000 cells per well. For measurement of in-
tracellular cAMP accumulation, cells were incubated with GloSensor
cAMP reagent for two hours and then stimulated with different CTR
ligands. The cAMP signal was measured by the plate reader Mithras
LB940 (2013, Berthold Technologies).
3. Results and discussion
3.1. Synthesis of the control compound SUN-B8155 and its derivatives
The synthetic routes for synthesizing SUN-B8155 and its derivatives
are described in Fig. 1. Hydroxylamine hydrochloride reacted with two
equivalents of diketene in the presence of triethylamine, resulting in the
formation of 5-acetyl-1, 6-dihydroxy-4-methyl-2(1H)-pyridone (1) in
only 26% yield [37,38]. When a mixture of water and acetonitrile (2:1
v/v) was used as solvent, the reaction yield was increased to 50%.
Different bases (such as pyridine, LiOH, NaOH, K2CO3, etc.) have been
used to replace triethylamine, but none improved the yield. Ketone 1
and the substituted anilines were heated at reflux in EtOH [39] to afford
SUN-B8155 and its derivatives (S8155-1, S8155-2, S8155-4 to S8155-
9), respectively. Catalytic reduction of 1 with Raney nickel in MeOH
gave 5-acetyl-6-hydroxy-4-methyl-2(1H)-pyridone (2) in 80% yield.
Under the same reaction conditions used for the preparation of SUN-
B8155 and its derivatives, 2 was reacted with one equivalent amount of
O-phenylenediamine to generate S8155-3 in 60% yield (Fig. 1). The
structural characterization and purity of all the compounds described
above were confirmed by their spectral properties (Supplemental file).
Detailed information can be seen in “2.1 Chemical synthesis” in the
section “Materials and Methods”.
2.4. BRET assay
For measurement of β-arrestin-2 recruitment, HEK293 cells were co-
transfected with plasmids encoding CTR-YFP and Luc-β-arrestin-2 for
48 h. After 12 h of starvation (with low glucose medium culture), cells
were harvested and washed at least three times with PBS, and then cells
were stimulated with different CTR ligands for 10 min at 37 °C.
Subsequently, transfected cells were incubated with Coelenterazine h
(Promega S2011, final concentration of 5 μM) at rt, and two different
light emissions were used for measurement (480 nm for luciferase and
530 nm for yellow fluorescent protein). All the BRET measurements
were performed by Mithras LB940 (2013, Berthold Technologies), and
the signal was determined by calculating the ratio of light intensity
emitted by yellow fluorescent protein over the intensity emitted by
luciferase.
2.5. Bias property quantification
3.2. Biological characterization of SUN-B8155 and its derivatives
The bias of CTR ligands are quantified according to previous studies
(shown as Eq. (1) at the end of the paragraph) [40,48], Both in cAMP
assay and arrestin recruitment BRET assay, concentration-dependent
response curves were used to determine the EC50 and Emax using
Graphpad. The P1 and P2 denote signaling through GS or β-arrestin-2
activity respectively. “lig” is the abbreviation used for “ligand”, which
denotes the 9 SUN8155 derivatives. “ref” is the abbreviation used for
“reference”, which denotes the compound SUN-B8155. β is the bias
factor, which is calculated as the logarithm of the ratio of intrinsic re-
lative activities for a ligand at two different assays compared with a
reference agonist. A bias factor of 1 between two pathways means that a
ligand is 10 times better for one pathway over the other pathway
compared with the reference balanced agonist [40].
Calcitonin, a 32-amino acid peptide hormone secreted mainly from
the thyroid gland, plays an important role in maintaining bone home-
ostasis [42-45]. Previous research showed that SUN-B8155 is a pyridine
derivative that mimic the biological activities of CT, acting via the CTR
[20]. SUN-B8155 elevates cAMP accumulation in human and rat cells
and this effect was blocked by CTR antagonist (sCT 8-32). Here, we
determined the ability of SUN-B8155 to activate Gs protein signalling
and the recruitment of β-arrestin-2 in CTR-overexpressing HEK293
cells. The EC50 value of SUN-B8155 for eliciting intracellular cAMP
accumulation levels through human CTR was determined to be
17.3
2 µM, whereas the EC50 of SUN-B8155 for recruitment of β-
arrestin-2 was 80.7
26.5 µM (Table 1 and Fig. 2). Therefore, the
synthesised SUN-B8155 elicits both Gs and β-arrestin-2 mediated sig-
nalling downstream of CTR activation.
E max,P1 EC50, P2
EC50, P1 E max,P2
E max,P2 EC50, P1
EC50, P2 E max,P1
=
lig
ref
(1)
We then measured the ability of SUN-B8155 derivatives (S8155-1 to
S8155-9) to promote cAMP accumulation and β-arrestin-2 recruitment
in CTR-transfected cells and compared them to that of SUN-B8155
(Table 1 and Fig. 3). Firstly, elimination of the N-hydroxyl group from
the pyridone ring abolished the activity of S8155-3 in cAMP accumu-
lation and decreased the EC50 of β-arrestin-2 recruitment by at least 6-
fold (Fig. 2B and Table 1). This result indicated that an H-bond or polar
2.6. Transwell® assay
Transwell assays were performed by using transwell chambers with
an 8.0 μm membrane. Briefly, MCF-7 cells were transfected with CTR-
YFP using PEI (Polysciences) according to the manufacturer’s
3

S. Zhao, et al.
BioorganicChemistry96(2020)103596
Fig. 1. Synthetic route for SUN-B8155 and its derivatives.
interaction of SUN-B8155 derivatives formed by N-OH with CTR is a
key interaction in the mediation of both the binding of the compound to
the receptor and its activation of the Gs signalling pathway.
that this could be due to the effect of substitution of the NH2 group of
the phenyl ring of SUN-B8155 by the CH3 group; which may result in
increased hydrophobic interactions within the receptor site.
Taken together, by synthesizing and screening the nine SUN-B8155
derivatives, we have acquired a compound with slightly improved Gs
signalling (S8155-7), and a compound with significant β-arrestin-2
biased properties (S8155-4). How the differences in chemical structures
determine the bias properties of S8155-4 and S8155-7 still waits for
further structural elucidations.
Secondly, moving the NH2 group of SUN-B8155 from the ortho to
meta or para positions in S8155-2 or S8155-4, respectively, greatly
improved their EC50 in recruiting β-arrestin-2, but has no significant
effect on their EC50 in cAMP accumulation. In particular, S8155-4 has
an EC50 of 7.6
2.5 µM and only 10% of the Rmax in cAMP accu-
mulation compared to SUN-B8155. Consistently, the β values for
S8155-2 and S8155-4 are calculated as −1.38 and −2.14 respectively,
according to Eq. (1). This result suggested that the property of S8155-4
activating the β-arrestin-2 pathway is 100 folds stronger than the Gs
pathway, taking SUN-B8155 as the balanced ligand.
3.3. Effects of SUN-B8155 derivatives on tumour cell invasion
Previous studies report that calcitonin and its analogues are in-
volved in cancer cell growth. For example, MCF-7 and other several
cancer cell lines contain specific high-affinity receptors for calcitonin
and a calcitonin-responsive adenylate cyclase, which have been char-
acterized with the aid of salmon, human calcitonin and their analogues
[10,15]. In T 47D breast cancer cell line, low doses of salmon calcitonin
Thirdly, among the nine newly synthesised SUN-B8155 analogues,
S8155-7 is the only compound showing slightly increased Gs bias
property compared to SUN-B8155, with a β value of 0.21 as calculated
by Eq. (1) [32,40,41]. It also exhibits an increase in its EC50 value by
approximately 1.7-fold without significant effect on Rmax. It is possible
Table 1
Bias properties of the 9 derivatives of SUN-B8155.
Ligand
Gs
β-arrestin-2
Rmax (%)
β
Bias
Rmax (%)
EC50 (μM)
EC50 (μM)
SUN-B8155
S8155-1
S8155-2
S8155-3
S8155-4
S8155-5
S8155-6
S8155-7
S8155-8
S8155-9
100.00
103.83
99.50
–
17.30
477.48
112.45
–
2.05
100.00
114.45
136.93
108.32
80.71
324.29
29.78
538.01
7.59
26.59
106.81
9.81
177.25
0.000
38.94
21.16
56.58
13.32
28.49
34.09
39.01
−0.879
−1.385
–
β-arrestin-2
β-arrestin-2
β-arrestin-2
β-arrestin-2
β-arrestin-2
β-arrestin-2
Gs
10.13
90.90
39.19
97.14
38.91
101.24
1.71
6.75
3.90
4.09
6.51
20.87
25.60
40.83
58.65
11.59
56.57
18.51
3.03
89.28
81.91
75.69
65.59
138.70
96.50
22.23
23.84
15.65
9.41
2.50
−2.142
−0.304
−0.908
0.210
4.84
6.95
1.37
6.70
2.19
85.23
65.38
59.24
105.33
41.08
28.07
21.53
19.51
30.78
13.53
55.15
63.55
−0.951
−0.302
β-arrestin-2
β-arrestin-2
Annotation. Rmax (%) represents the ratio of each derivative’s Emax to that of SUN-B8155. β is called bias factor. This bias factor is an estimate for the molecular
efficacy of Gs pathway or β-arrestin-2 pathway on a logarithmic scale. If β > 0.000, it means the bias property of the Gs pathway; if β < 0.000, it indicates the bias
property of the β-arrestin-2 pathway. “–” is a null value which is not be able to be determined.
4

S. Zhao, et al.
BioorganicChemistry96(2020)103596
Fig. 2. Measurement of cAMP accumu-
lation and β-arrestin-2 recruitment in
response to S8155 1-9 stimulation. (A-
B) Effects of S8155 1-9 on cAMP accu-
mulation in HEK293 cells co-trans-
fected with CTR-YFP and GloSensor.
“RLU” mean relative light unit which
measured the level of cAMP accumula-
tion in living cells. (C-D) Effects of
S8155 1-9 on β-arrestin-2 recruitment
in HEK293 cells co-transfected with
CTR-YFP and Luc-β-arrestin-2. Data are
shown as mean
presentative of
SEM and re-
3
individual experi-
ments. The BRET Ratio was determined
by dividing the intensity of acceptor
(YFP) luminescence emission at 530 nm
by the intensity of donor (luciferase)
luminescence emission at 480 nm.
study the biased signalling of CTR in contribution to its inhibition role
in cancer progression in MCF-7 cells. When we treated MCF-7 cells with
1 µM sCT, the tumour invasion ability significantly decreased in the
Transwell® assay (Fig. 3). The application of 10 µM SUN-B8155 has a
much stronger effect on the invasion of MCF-7, as does the application
of 10 µM S8155-4 or 10 µM S8155-7. Importantly, the application of the
CTR antagonist sCT (8-32) significantly blocked the ability of SUN-
B8155, S8155-4 and S8155-7 to inhibit MCF-7 cell migration (Fig. 3).
Therefore, we speculate that the effects of these compounds on MCF-7
cell invasion are partially due to the activation of CTR. However, fur-
ther in vivo studies are still needed to explain the detailed mechanism
of how Gs or β-arrestin-2 mediated pathways contribute to cancer cell
migration inhibition downstream of CTR.
4. Conclusion
SUN-B8155 activated both the Gs and arrestin pathways through its
engagement with CTR, which is dependent on the hydroxyl group at-
taching to the pyridine amide of SUN-B8155. Analogues of SUN-B8155
can act as agonists of CTR through the Gs or arrestin pathway at dif-
ferent levels. S8155-7 improved Gs signalling, whereas S8155-4 is β-
arrestin-2-biased. S8155-4 and S8155-7 inhibited breast cancer cell
migration through CTR agonism.
Fig. 3. Effects of different CTR ligands on tumor cell invasion. Transwell assays
were performed on MCF-7 cells to detect the effects of different CTR ligands.
sCT, salmon calcitonin (1 μM); sCT 8-32 (1 μM); SUN-B8155 and S8155 deri-
vatives were applied at a concentration of 10 μM. **p < 0.005, ***p < 0.0001,
inhibition of tumor cell migration by different ligands were compared with that
of vehicle. ##p < 0.005, inhibition of tumor cell migration by treatment with
sCT8-32 was compared with no treatment.
Our research provides useful chemical tools for studying CTR-
mediated signaling pathways. Future work will involve investigating
effects of these compounds in different CTR-mediated patho-physiolo-
gical processes as well as structural determination of the CTR in com-
plex with these small chemical probes. This will facilitate therapeutic
development targeting CT receptors.
initially stimulated cell growth and then followed by an inhibitory ef-
fect for cell proliferation and migration, which occurred during the log
phase of growth [11]. In DU145 prostate cancer cells, calcitonin in-
duced the inhibition of mitogen-activated kinase and prevented pros-
tate cancer progressing [12]. However, how activation of different CTR
downstream effectors participated in the function of CTR in tumor in-
hibition in these different cellular contexts remain elusive.
Funding
Here, we have acquired the S8155-7, a compound that slightly ac-
tivates more Gs signalling downstream of CTR, as well as a compound
S8155-4 with significant β-arrestin-2 bias. These compounds could
serve as tools for CTR functional studies. We then used these tools to
This work was financially supported by the National Key Basic
Research Program of China Grant (2018YFC1003600 to J.-P.S.), the
National Science Fund for Distinguished Young Scholars Grants
(81773704 to J.-P.S.), the National Science Fund for Excellent Young
5

S. Zhao, et al.
BioorganicChemistry96(2020)103596
Scholars Grant (81822008 to X.Y.; 81922071 to Y.Z.), the National
Science Foundation of China (21272029 to X. C.), and Zhejiang
Province National Science Fund for Excellent Young Scholars
(LR19H310001 to Y.Z.).
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Author contributions
[18] S. Gydesen, K.V. Andreassen, S.T. Hjuler, L.I. Hellgren, M.A. Karsdal, K. Henriksen,
Optimization of tolerability and efficacy of the novel dual amylin and calcitonin
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10.1152/ajpendo.00419.2016.
Professors JP S, X C and Y Z planned the research. S Z, YM W and XY
J finished the synthesis of the 9 derivatives of SUN-B8155 under the
guidance of professor X C. SC G and Z G performed the experiment to
test the function of the 9 derivatives as agonist to CTR under the gui-
dance of professor JP S and X Y. Professor Y Z gave guidance to analyze
the data. SC G, Z G and C Y wrote the paper. Professor X C helped polish
the manuscript.
[19] S. Gydesen, S.T. Hjuler, Z. Freving, K.V. Andreassen, N. Sonne, L.I. Hellgren,
M.A. Karsdal, K. Henriksen, A novel dual amylin and calcitonin receptor agonist,
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K. Magota, M. Saito, S. Tanaka, S. Oikawa, Discovery of a non-peptide small mo-
lecule that selectively mimics the biological actions of calcitonin, Biochim. Biophys.
Acta, Mol. Cell. Biol. Lipids 1526 (2001) 183–190, https://doi.org/10.1016/S0304-
4165(01)00125-8.
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Appendix A. Supplementary material
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