A novel Apigenin derivative suppresses renal cell carcinoma via directly inhibiting wild-type and mutant MET

Article abstract of DOI:10.1016/j.bcp.2021.114620

MET, the receptor of hepatocyte growth factor (HGF), is a driving factor in renal cell carcinoma (RCC) and also a proven drug target for cancer treatment. To improve the activity and to investigate the mechanisms of action of Apigenin (APG), novel derivatives of APG with improved properties were synthesized and their activities against Caki-1 human renal cancer cell line were evaluated. It was found that compound 15e exhibited excellent potency against the growth of multiple RCC cell lines including Caki-1, Caki-2 and ACHN and is superior to APG and Crizotinib. Subsequent investigations demonstrated that compound 15e can inhibit Caki-1 cell proliferation, migration and invasion. Mechanistically, 15e directly targeted the MET kinase domain, decreased its auto-phosphorylation at Y1234/Y1235 and inhibited its kinase activity and downstream signaling. Importantly, 15e had inhibitory activity against mutant MET V1238I and Y1248H which were resistant to approved MET inhibitors Cabozantinib, Crizotinib or Capmatinib. In vivo tumor graft study confirmed that 15e repressed RCC growth through inhibition of MET activation. These results indicate that compound 15e has the potential to be developed as a treatment for RCC, and especially against drug-resistant MET mutations.

Full text of DOI:10.1016/j.bcp.2021.114620

Biochemical Pharmacology 190 (2021) 114620
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
A novel Apigenin derivative suppresses renal cell carcinoma via directly
inhibiting wild-type and mutant MET
a
,
1
a
,
b
,
1
c
a
,
d
c
c
c
Jing Li , Guishan Tan
, Yabo Cai , Ruihuan Liu , Xiaolin Xiong , Baohua Gu , Wei He ,
c
c
a
c
c
e
a
Bing Liu , Qingyun Ren , Jianping Wu , Bo Chi , Hang Zhang , Yanzhong Zhao , Yangrui Xu ,
Zhenxing Zou , Fenghua Kang , Kangping Xu
a
a
a,*
a
Xiangya School of Pharmaceutical Sciences, Central South University, Changsha 410013, China
Xiangya Hospital of Central South University, Changsha 410008, China
b
c
State Key Laboratory of Anti-Infective Drug Development, Sunshine Lake Pharma Co. Ltd, Dongguan 523871, China
Zhuzhou Qianjin Pharmaceutical Co. Ltd, Zhuzhou, 412007, China
d
e
The Third Xiangya Hospital, Central South University, Changsha 410013, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
MET, the receptor of hepatocyte growth factor (HGF), is a driving factor in renal cell carcinoma (RCC) and also a
proven drug target for cancer treatment. To improve the activity and to investigate the mechanisms of action of
Apigenin (APG), novel derivatives of APG with improved properties were synthesized and their activities against
Caki-1 human renal cancer cell line were evaluated. It was found that compound 15e exhibited excellent potency
against the growth of multiple RCC cell lines including Caki-1, Caki-2 and ACHN and is superior to APG and
Crizotinib. Subsequent investigations demonstrated that compound 15e can inhibit Caki-1 cell proliferation,
migration and invasion. Mechanistically, 15e directly targeted the MET kinase domain, decreased its auto-
phosphorylation at Y1234/Y1235 and inhibited its kinase activity and downstream signaling. Importantly,
Apigenin derivatives
Renal cell carcinoma
MET
MET downstream signaling
Drug-resistant MET mutations
1
5e had inhibitory activity against mutant MET V1238I and Y1248H which were resistant to approved MET
inhibitors Cabozantinib, Crizotinib or Capmatinib. In vivo tumor graft study confirmed that 15e repressed RCC
growth through inhibition of MET activation. These results indicate that compound 15e has the potential to be
developed as a treatment for RCC, and especially against drug-resistant MET mutations.
1
. Introduction
Renal cell carcinoma (RCC), a major form of kidney cancer, is one of
for clear cell RCC [4], receptor tyrosine kinase (RTK) MET activation is
also an important factor in renal cancer progression [5]. MET, the re-
ceptor for hepatocyte growth factor (HGF), is normally activated by HGF
binding but can also be activated by mutations and gene amplification
and overexpression [6]. During the process of MET activation, auto-
phosphorylation at Y1234 and Y1235 after MET dimerization is crit-
ical and an indicator for its kinase activity [7]. Once activated, MET
transduces signals down toward RTK classical pathways including PI3K-
Akt, Ras-Raf-MEK-Erk and Stat3 to regulate cell survival, growth, pro-
liferation, and mobility [8]. In RCC, MET is activated by mutation and
upregulated expression [9,10]. Mutations such as L1213V, V1238I,
the most common cancers worldwide [1]. Clear cell RCC [2] and
papillary RCC [3] account for approximately 75% of RCC and 15% of all
kidney cancers respectively [1]. Although nephrectomy is an effective
therapy for most patients with localized disease, metastasis occurs in
many patients ultimately experience disease relapse [4]. There are still
tremendous unmet medical needs for RCC therapy.
In addition to inactivation of Von Hippel-Lindau disease tumor
suppressor (VHL) which is a hallmark and an important driving factor
Abbreviations: 3-D, 3-dimensional; APG, Apigenin; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HGF, hepatocyte growth factor; HRP, horseradish
peroxidase; hrs, hours; IC50, half maximal inhibitory concentration; IL3, interleukin 3; mTOR, mechanistic target of rapamycin pathways; PAINS, pan-assay inter-
ference compounds; RCC, renal cell carcinoma; RTK, receptor tyrosine kinase; TPR, translocated promoter region; VHL, Von Hippel-Lindau disease tumor suppressor;
WT, wild-type.
*
Corresponding author.
E-mail address: xukp395@csu.edu.cn (K. Xu).
Jing Li and Guishan Tan contributed equally to this work.
1
https://doi.org/10.1016/j.bcp.2021.114620
Received 23 February 2021; Received in revised form 20 May 2021; Accepted 20 May 2021
Available online 24 May 2021
0
006-2952/© 2021 Elsevier Inc. All rights reserved.

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
D1246N, Y1248H, or M1268T, were found in hereditary and sporadic
papillary RCC [9,10]. These mutant MET display constitutive tyrosine
phosphorylation [11], and are resistant to approved MET kinase inhib-
itor Cabozantinib, Capmatinib or Crizotinib [12–15]. In fact, mutant
MET was more easily activated by HGF and stay activated for longer
time than wild-type (WT) MET [16]. These mutations, coupled with the
high expression of HGF in the kidney might explain why patients with
germline MET mutation more likely have kidney cancer [5]. In vitro and
in vivo studies demonstrated that the inactivation of VHL might increase
MET expression in clear cell RCC [17–19]. Furthermore, phosphorylated
MET was higher in RCC tumors [20,21], and indeed MET phosphory-
lation was correlated with greater proliferation index, greater tumor
diameter, and worse cause-specific survival [21].
resistant to current drugs. In addition, in an in vivo mice tumor model,
15e repressed RCC growth through inhibition of MET activation. In
summary, we identified an APG derivative, 15e, which has the potential
to be developed against RCC via a mechanism of directly targeting MET.
2. Materials and methods
2.1. Materials in chemistry
Solvents and regents were commercially available and used without
further purification, unless otherwise stated. All reactions were moni-
tored by thin layer chromatography (TLC) on silica gel GF254 (0.25 mm,
Qingdao Ocean Chemical Ltd., China). Flash chromatography was per-
formed using silica gel 60 (230–400 mesh, Qingdao Ocean Chemical
Ltd., China). The purities of all target compounds were estimated by
Waters 1525 Binary HPLC Pump. 1H NMR and C NMR spectra were
recorded with an Agilent NMR Inova 400 or 600 spectrometer with TMS
as the internal standard. All chemical shift d was given in parts per
million (ppm). Mass spectra were recorded on an Agilent HPLC1260-
MS6120 with ESI source as ionization, respectively. Chemical raw ma-
terials (1, 2 and 9) were provided by Bidepharm, Shanghai, China.
Apigenin and Crizotinib were purchased from MedChemExpress (HY-
N1201, China) and Selleck (S1068, China).
APG is a natural flavone, and is abundant in vegetables, fruits and
herbs. In addition to anti-inflammation and anti-oxidation, the most
widely studied function of APG is its anti-cancer activity against a va-
riety of tumors including skin, breast, prostate, digestive tract, and
certain hematological malignancies [22]. At the molecular level, APG
was indicated to target a variety of proteins and signaling pathways
directly or indirectly. A number of studies have illustrated that APG can
inhibit the signaling pathways such as PI3K-Akt, JAK-Stat, WNT-β-cat-
enin and NF-κB [22], which are all involved in carcinogenesis and
metastasis. APG can also directly bind to hnRNPA2 and inhibit its
dimerization and function [23]. PDGFR and Src kinase were also
thought to be targets of APG [24–26]. However, the further develop-
ment of APG for cancer therapy has been challenging since it has sub-
optimal efficacy and bioavailability [22]. Nevertheless, efforts and
progress have been made recently [27,28].
13
2.2. Synthetic route
The general synthetic routes of novel 5,7-dihydroxy-2-(6-hydroxy-
′
[1,1 -biphenyl]-3-yl)-4H-chromen-4-one derivatives were illustrated in
Whether APG has direct inhibitory activity against MET has not been
conclusive. One study reported that high concentrations of APG
inhibited MET phosphorylation [29]. In addition, other compounds with
structural similarities to APG, such as Rutin, Isoquercitrin and Nobiletin,
were reported to inhibit MET phosphorylation [30–32]. Yet another
study indicated that APG had no effect on phosphorylated MET in MDA-
MB-231 cells stimulated by HGF [33]. These inconclusive results could
be due to the lack of potency of APG.
Scheme 1 [37,38]. The commercially material 1-(2,4-dihydroxy-6-
methoxyphenyl) ethan-1-one (1) was treated with 3-iodo-4-methoxy-
benzaldehyde (2) in potassium hydroxide solution to give (E)-1-(2-hy-
droxy-4,6-dimethoxyphenyl)-3-(3-iodo-4-methoxyphenyl)prop-2-en-1-
one (3), which was reacted with iodine to obtain compound 4. Subse-
quently, compounds 6a-e were prepared through Suzuki-Miyaura
coupling reaction of compound 4 with boron reagents. Then, two-step
demethylative reactions were performed to achieve the target prod-
ucts (8).
Anti-tumor effects of APG on RCC has not been widely studied. APG
was found to have anti-RCC activity in vitro and in vivo through inducing
cell cycle arrest and apoptosis [34]. Other APG-like compounds such as
Vitexin and Scutellarin were shown to suppress RCC through inhibition
of P13K-AKT-mTOR pathway [35,36]. To improve the activity of APG
and to further study its anti-RCC activity, we modified the peripheral
substituents of APG while keeping its core ring structure unchanged.
Hence, a series of APG derivatives were synthesized and screened for
their activity on a RCC cell line Caki-1 which carries a Cabozantinib-
resistant V1238I mutation in MET [15]. We discovered that compound
The general synthetic routes of novel 5-hydroxy-2-(4-hydroxy-
phenyl)-7-methoxy-8-phenyl-4H-chromen-4-one derivatives were illus-
trated in Scheme 2 [39,40]. The commercially material 1-(2-hydroxy-
4,6-dimethoxyphenyl)ethan-1-one (9) was treated with iodine to obtain
compound 10, which was reacted with 4-(benzyloxy)benzaldehyde (11)
in potassium hydroxide solution to give (E)-3-(4-(benzyloxy)phenyl)-1-
(2-hydroxy-3-iodo-4,6-dimethoxyphenyl)prop-2-en-1-one (12). Next, a
cyclization reaction was proceeded to achieve 2-(4-(benzyloxy)phenyl)-
8-iodo-5,7-dimethoxy-4H-chromen-4-one (13). Subsequently, com-
pounds 15a-k were prepared through Suzuki-Miyaura coupling and
deprotection reactions.
1
5e is most potent in inhibiting Caki-1 cell proliferation among APG and
its derivatives and is superior to Crizotinib and Cabozantinib. Further
mechanism studies indicated that 15e directly targeted MET, and
inhibited its kinase activity and downstream signaling. Importantly, 15e
inhibited mutant MET with V1238I and Y1248H mutations that are
◦
◦
◦
Scheme 1. Reagents and conditions: i) KOH (30 wt%), EtOH; ii) I
2
, DMSO, 110 C; iii) Pd(PPh ) , Cs CO , DMF/H O, 110 C, iv) AlCl , 85 C; v) BBr , rt.
3 4 2 3 2 3 3
2

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
◦ ◦ ◦
2 3 2 3 4 2 3 2 3
Scheme 2. Reagents and conditions: i) I , HNO , EtOH; ii) KOH (30 wt%), EtOH; iii) I , DMSO, 110 C; iv) Pd(PPh ) , Cs CO , DMF/H O, 110 C; AlCl , 85 C;
3
BBr , rt.
2
.3. Cell culture
Cell lines in this investigation include following: human RCC cell line
2.7. Phospho-RTK assays
Phosphorylated RTKs were measured with the Human Phospho-RTK
Array Kit (ARY001B) from R&D Systems according to the manufac-
turer’s instructions. Briefly, 80% confluence Caki-1 cells in 10-cm dishes
were treated with the indicated concentration of compound for 24 hrs.
Cells were lysed with lysis buffer and protein concentration of the lysate
was measured by BCA kit (ThermoFisher Scientific, 23225). Aliquots of
Caki-1 (ATCC, HTB-46), Caki-2 (ATCC, HTB-47), and ACHN (ATCC,
CRL-1611); human lung cancer cell line NCI-H441 (ATCC, HTB-174)
and NCI-H446 (ATCC, HTB-171); human gastric carcinoma cell line
SNU-5 (ATCC, CRL-5973); human breast cancer cell line MCF7 (ATCC,
CRL-3435) and MDA-MB-231 (ATCC, HTB-26); human melanoma cell
line SK-MEL-28 (ATCC, HTB-72); mouse pro-B cell line Ba/F3 (DMSZ,
CM-1517); mouse embryonic fibroblast cell line NIH-3T3 (ATCC, CRL-
300 g lysate was added to each well and incubated at 4℃ overnight on
μ
a shaker. After washes, anti-phospho-tyrosine-HRP detection antibody
within the kit was added to each well and incubated at room tempera-
ture for 2 hrs on a shaker. Then the arrays were incubated with Chemi
Reagent Mix and photographed with chemiluminescence system (Bio-
Rad, ChemiDoc XRS+). Phospho-RTK assay was repeated two times.
1
658); human embryonic kidney cell 293T (ATCC, CRL-3216). For cell
culture, SK-MEL-28, MDA-MB-231, Ba/F3 and SNU-5 cells were incu-
bated in RPMI 1640 (HyClone, SH30809.01B) and the other cell lines
◦
were cultured in DMEM (HyClone, SH30243.01B) at 37 C with 5% CO
2
.
RPMI 1640 and DMEM were supplemented with 10%(v/v) fetal bovine
serum (Gibco, 10091148) and 1%(v/v) penicillin–streptomycin
2
.8. Apoptosis assay
(
HyClone, SV30010). All cell passages were kept under 20.
5
2
× 10 Caki-1 cells seeded into 6-well dishes were incubated for 24
hrs before indicated concentrations of compounds were added for
another 24 hrs incubation. Then, the cells were digested and suspended,
washed with PBS twice and stained with Annexin V-FITC and PI staining
2
.4. Cell viability assay
Cell viability assay kit was purchased from Dojindo Molecular
◦
buffer (Dojindo Molecular Technologies, AD10) for 30 min at 37 C. The
Technologies (CK04). The assay was performed according to the tech-
3
resulting samples were analyzed by flow cytometry using Beckman
Cytoflox. Apoptosis assay was repeated three times.
nical manual. Briefly, 6 × 10 cells were plated into 96-well dishes and
incubated with different concentrations of compounds for 72 h (hrs).
1
0% (v/v) final concentration of CCK-8 buffer was then added and the
cell were further cultured for 1–2 h before measurement of absorbance
at 450 nm using a microplate reader. This assay was repeated three
times.
2.9. Cell cycle assay
5
2 × 10 Caki-1 cells were seeded into 6-well dishes and incubated for
2
4 hrs before culture medium was replaced with fresh medium con-
taining compounds and incubated for another 24 hrs. The cells were
then harvested, washed with ice-cold PBS and fixed with ice-cold 70%
2
.5. Colony formation assay
◦
ethanol at 4 C for 12 hrs before stained with PI staining buffer (Beyo-
3
◦
1
× 10 cells were plated into 12-well dishes and cultured at 37 C
◦
time, C1052) for 30 min at 37 C. The resulting samples were analyzed
with 5% CO
2
for 24 hrs before the indicated dose of chemical compound
by flow cytometry using Beckman Cytoflox. Cell cycle assay was
repeated three times.
was added and the cells were incubated for about 7 days before the a
colony was stained using 0.5% crystal violet solution (Beyotime,
C0121). Colony formation assay was repeated three times.
2
.10. Immunoblot
5
2
.6. Growth of Caki-1 cell 3-dimensional (3-D) microsphere
2 × 10 Cells were seeded in 6-well dishes overnight prior to treat-
ment with the indicated concentrations of compound. Cells were quickly
3
4
× 10 Caki-1 cells per well were seeded in U-bottom 96-well dishes
lysed by adding 200
μL 2 × SDS buffer, and then collected and incubated
◦
(
ThermoFisher Scientific, 168136) and cultured for 4 days to form 3-D
at 95 C for 5 min. The proteins were separated on SDS-PAGE (sodium
dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred to
polyvinylidene fluoride membranes (BioRad, 1620177). The mem-
branes were blocked with 5% BSA/PBST buffer at room temperature for
microspheres. 15e or APG (MCE, HY-N1201) were then added and the
microsphere were further incubated for another 8 days before being
photographed under microscopy (Nikon, Ts2R-FL) at 100×. This
experiment was repeated three times.
◦
1 h and then incubated at 4 C overnight with primary antibodies in 5%
3

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
BSA/TBST buffer with gentle shaking. Primary antibodies in the
research were purchased from Cell Signaling Technologies, including
MET (#8198), p-MET-Y1234/Y1235 (#3077), Akt (#2920), p-Akt-S473
2.14. Trans-well migration assay
4
7 × 10 cells in FBS-free DMEM with different concentrations of
(
#4060), p-Akt-T308 (#4056), Stat3 (#9139), p-Stat3-Y705 (#9145),
compounds were seeded into the upper apartment of 8
μ
M pores trans-
Erk1/2 (#4696), p-Erk1/2-T202/Y204 (#4370), PRAS40 (#2691), p-
PRAS40-T246 (#2997), mTOR (#2983), p-mTOR-S2448 (#5536), p70-
S6K (#2708), and p-p70-S6K-T389 (#9234). All primary antibodies
were 1:1000 diluted in PBST buffer (PBS + 0.5%(v/v) Tween-20) with
well device (Corning, 3422) containing 500 L 10%-FBS-containing
μ
DMEM with or without 15 ng/mL HGF (R&D Systems, 294-HG) in the
◦
lower apartment. After the device was incubated at 37 C with 5% CO
2
for 16 hrs, cells in upper wells were completely removed with cotton
swabs. Cells that had migrated to the lower surface of the filters were
fixed with methanol and stained with crystal violet (Beyotime, C0121).
Finally cells migrated to the lower surface of the filters were photo-
graphed under microscopy at 100×. This experiment was repeated three
times.
5
% BSA. After the primary antibodies incubation, the membranes were
washed with PBST three times and incubated with appropriate
peroxidase-conjugated secondary antibodies (anti-IgG-HRP, 1:5000,
CST, #7074, or #7076) for 1 h at room temperature and washed again.
Protein bands were visualized using enhanced ECL western blotting
detection reagents (ThermoFisher Scientific, SuperSignal West Dura,
3
4076) followed by capturing photos with chemiluminescence system
(
BioRad, ChemiDoc XRS+). Bands in these photos were quantified
2.15. Molecular docking
through measuring gray value with Image J (NIH). Each immunoblot
was repeated at least three times.
The crystallography structure of MET was downloaded from the
Protein Data Bank (http://www.rcsb.org, PDB code 3F66). All water
molecules were deleted. The protein structure was prepared using the
Schro ¨d inger’s Protein Preparation Wizard (Schr o¨ dinger Release 2018–1:
Schr o¨ dinger Suite 2018–1 Protein Preparation Wizard; Epik,
Schr o¨ dinger, LLC, New York, NY, 2016; Impact, Schr o¨ dinger, LLC, New
York, NY, 2016; Prime, Schr o¨ dinger, LLC, New York, NY, 2018.) in
Maestro version 11.5. The molecular structure of 15e was generated
using Schro ¨d inger’s 3D Builder tool and the ligand was prepared using
the Schro ¨d inger’s LigPrep tool (Schr o¨ dinger Release 2018–1: LigPrep,
Schr o¨ dinger, LLC, New York, NY, 2018.). The receptor grids for the
docking procedure were generated using the native ligand of the crystal
structure as the centroid of the grid box. 15e was docked into the pre-
pared protein using Glide (Schr o¨ dinger Release 2018–1: Glide,
Schr o¨ dinger, LLC, New York, NY, 2018.) in the extra precision (XP)
mode. The number of poses per ligand to include in the postdocking
minimization step was set to 100, and the maximal number of docking
poses output for the ligand was set to 20.
2
.11. Plasmids, siRNA and transfection
Service of gene synthesis, gene clone and mutation, and siRNA was all
purchased from Synbio Technologies (Suzhou, China). cDNA of TPR-MET,
and WT or mutant MET (isoform 2) were inserted into vector pLVX-IRES-
Puro (Takara, 631253) and pCDNA3.1 (ThermoFisher Scientific, V790-20)
′
respectively. The sequences of si-MET were 5 -CAUCCAGAAUGUCAUU-
′
′
′
CUAdTdT-3 and 5 -UAGAAUGACAUUCUGGAUGdTdT-3 ; The sequences
′
of si-NC (siRNA negative control) were 5 -UUCUCCGAACGUGU-
′
′
′
CACGUdTdT-3 , and 5 -ACGUGACACGUUCGGAGAAdTdT-3 . 80%
confluence NIH-3T3 cells or Caki-1 cells in 6-well dishes were transfected
with1
μ
gMETplasmidor60pmolsiRNArespectivelybyusinglipofectamine
3000 (ThermoFisher Scientific, L3000008). 24 hrs later, cells were digested
5
andsuspended, and1× 10 NIH-3T3cellsperwellwereseededinto24-well
dishesandtreatedwith15efor24hrsbeforecellswerelysedwith100
μ
L2×
SDS for immunoblot analysis. si-RNA transfected Caki-1 cells were seeded
into 96-well dishes and treated with the indicated concentrations of 15e for
72 hrs before the cell viability was assessed by using CCK-8 assay kit.
2.16. In vivo xenograft tumor experiment
Four-week-old female athymic nude mice (BALB/c-nu) were pur-
chased from Hunan SJA Laboratory Animal Co., LTD. Mice were housed
under standard conditions with freedom to water and food, and were
subjected to a 12-hour light/dark cycle. All mice protocols were
approved by the Animal Ethics Committee at the Sunshine Lake Pharma
Co., LTD (Dongguan, China) and were in compliance with Chinese As-
2
.12. Lentivirus preparation and Ba/F3-TPR-MET stable cell-line
establishment
9
0% confluence 293 T cells in 6-well dishes were transfected with 2
μ
g lentivirus vector, 2
μ
g psPAX2 (Addgene, 12260) and 0.5 g pMD2.G
μ
(
Addgene, 12259) through lipofectamine 3000 (ThermoFisher Scienti-
fic, L3000008). 24 hrs later, culture medium was replaced with 2 mL
0%-FBS-containing DMEM, and the transfected cells continued to
being cultured for another 48 hrs until the culture medium was collected
and filtered with 0.22 M filter (Millipore, SLGP033RB). Filtered me-
sociation for Laboratory Animal Sciences guidelines. Caki-1 cell sus-
7
pension (1x10 cells in 200
μ
L of medium) was injected subcutaneously
2
into the right flank of each mouse on day 0. When the tumor volume
reached 200 mm3 the mice were randomized into vehicle control and
treatment groups (6 animals per group). 20 mg/kg 15e or APG (in 200
μ
dium with lentivirus then was used to incubate Ba/F3 cells for 24hrs.
Finally, infected Ba/F3 cells were cultured in medium without inter-
μ
L of vehicle solution (5% DMSO and 15% PEG400 in saline, pH 9) was
administered daily by intraperitoneal injection for 16 days while the
control group was treated with an equal volume of vehicle solution. The
tumor weight was measured at the termination of the experiment and
the body weight mouse was monitored throughout the experiment for
toxicity.
leukin 3 (IL3) for 9 days and then in medium with 0.5
Gibco, A1113803) for 4 days.
μg/mL puromycin
(
2
.13. Wound healing assay
5
2
× 10 Caki-1 cells were seeded per well in 6-well dishes and
2.17. Immunohistologic chemistry
incubated for 48 hrs until they reached 80%–90% confluence. A
confluent monolayer of Caki-1 cells was artificially wounded with a
micropipette tip, and the detached cells were washed with PBS and
treated with vehicle or indicated compounds in growing medium for 24
hrs. Images of the wounds were photographed at 0 and 24 hrs under
microscope (Nikon, Ts2R-FL) at 100×. This experiment was repeated
three times.
Tumor tissues were sectioned and immunohistochemically analyzed
for MET and p-MET-Y1234/Y1235 expression. Sections of the tumor
◦
tissues were incubated at 4 C overnight with the antibodies for MET and
p-MET-Y1234/Y1235, and detected using TMB Chromogen Solution
(Beyotime, P0211). The stained section was observed under an inverted
phase-contrast microscope and photographed.
4

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
2
.18. Data statistics
Table 1
IC50 of Caki-1 cells treated with APG and its derivatives.
Except phospho-RTK array, all experiments were performed at least
a
Compounds
IC50 ± SD (
μ
M)
three times, and representative data are shown. Statistical significance
APG
44.27 ± 2.02
4.43 ± 0.22
>50
(
p < 0.05) was assessed using Student’s t test or oneway ANOVA coupled
with Dunnett’s t test. Not significant (n.s.) indicated p > 0.05. All data
were represented as means ± SD.
Crizotinib
8a
8b
>50
8
8
7
7
c
28.57 ± 1.79
>50
d
d
e
3
. Results
>50
>50
3
.1. 1. Activity of APG derivatives against Caki-1 cell
15a
26.78 ± 1.49
5.66 ± 0.17
>50
1
1
1
1
5b
5c
5d
5e
The synthesized compounds (Fig. 1) were evaluated by CCK-8 assay,
>50
and were compared with Crizotinib as positive control. In CCK-8 assay,
Caki-1 cells were treated with APG or its derivatives for 72 hrs before
cell viability was measured. Then half maximal inhibitory concentration
0.49 ± 0.02
>50
15f
15g
>50
1
1
1
5h
5i
7.84 ± 0.99
11.02 ± 1.26
4.51 ± 0.08
24.64 ± 1.71
(
IC50) on cell viability was calculated and shown in Table 1, where the
synthesized compounds displayed moderate to high potency against
Caki-1 cell viability. Compound 15e with a para-acetylphenyl nonpolar
group was the most potent compound to affect Caki-1 cell viability.
Interestingly, the IC50 of 15e is 10 times better than Crizotinib (Table 1),
5j
15k
a
.
The average value of the three independent ex-
periments was expressed by mean ± SDs.
and is also better than the reported IC50 (14.5
approved RCC drug Cabozantinib [15].
μM in Caki-1 cell) for the
1
. In addition, 15e showed a remarkable improvement of anti-
proliferative activity over APG and Crizotinib in all three cell lines.
For later studies, Caki-1 cells were mainly used.
3
.2. In vitro activity of the APG derivative 15e against RCC cell growth
To re-confirm the ability of 15e in inhibiting Caki-1 cell growth, we
performed colony-formation and 3-D cell microsphere growth assays.
To further study and compare 15e and APG with Crizotinib for their
inhibition activity on RCC and to ascertain the observed effect with Caki-
can be extended to two other RCC cell lines, Caki-2 and ACHN, were
0
.625
or Crizotinib completely blocked the formation of colonies from Caki-1
cells, while 5 M APG had no effect on the colony-forming ability
Fig. 2B). Caki-1 cells formed 3-D microspheres after being cultured for
μ
M 15e clearly inhibited the formation of colonies and 5
μ
M 15e
1
further selected and investigated for their susceptibility to the com-
pounds. As shown in Fig. 2A, all three cell lines showed similar cell
viability profiles demonstrating that the inhibition is not limited to Caki-
μ
(
Fig. 1. The structures of APG and its novel synthesized derivatives. Structural changes are highlighted in red. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
5

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
Fig. 2. In vitro anti-tumor effects of APG derivative 15e. (A) Relative cell viability of Caki-1, Caki-2 and AHCN cells treated with 0.01–100
Crizotinib for 72 hrs. (B) Colony-forming ability of Caki-1 cells or (C) Growth of Caki-1 cell 3-D microsphere treated with vehicle, 0.625 M 15e, 5
or 5 M Crizotinib for 8 days respectively. The growth of Caki-1 cell 3-D microsphere was quantified by calculating the increment of sectional area of cell micro-
spheres. (D) Cell cycle analysis and (E) apoptosis assays of Caki-1 cells treated with indicated concentration of 15e or APG for 24 hrs. Data are expressed as the mean
SD, n = 3, *P < 0.05 versus Control. Not significant (n.s.) indicated P > 0.05.
μ
M 15e, APG or
μ
μ
M 15e, 5 M APG
μ
μ
±
6

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
4
days in a 96-well U-bottom dish (Fig. 2C, Day 0) and they can grow
cellular kinases [24,26,29,41]. Therefore, to explore the molecular
targets and mechanism of action of the anti-tumor activity of 15e against
Caki-1 renal cancer cell, we screened a panel of 49 different RTKs by
using a phospho-RTK array to detect which kinase is the target of 15e.
Specific RTK capture antibodies were spotted in duplicate on the array,
so when a cell lysate is applied to the array, RTKs, both phosphorylated
and un-phosphorylated, will be captured. A second anti-phospho-
tyrosine antibody conjugated to horseradish peroxidase (HRP) is then
applied to detect the presence and the abundance of that specific
phosphorylated kinase. As shown in Fig. 4A, lysates from Caki-1 cells
contained a variety of detectable phosphorylated RTK and can be
detected using this array, for example MET, EGFR, PDGFRβ and Ryk, at
bigger after another 8-day incubation (Fig. 2C, Day 8). When 15e or APG
was added during the 8-day incubation period, the growth of cell mi-
crospheres was significantly repressed by 5
not by APG (Fig. 2C).
μM 15e and Crizotinib but
Loss of cell viability measured by CCK8 can be due to cell death or
lack of cell proliferation. Hence, assays for apoptosis or cell cycle were
employed to check whether 15e could function with either of these two
aspects. When exponentially growing Caki-1 cells were incubated with
5
μ
M or 20
μ
M 15e, cells at G2/M increased indicating that the cell cycle
was blocked at this phase (Fig. 2D). Even at 20
μM, APG did not affect
the cell cycle, demonstrating the improvement in 15e activity. To assess
whether 15e is simply cytotoxic and causing cell death, apoptosis of
Caki-1 cells in the presence or absence of compounds was investigated
by flow cytometry. Neither 15e nor APG caused significant apoptosis
the experimental conditions. Treatment of Caki-1 cells with 5 M 15e for
μ
24 hrs, however, affected the amount of several phospho-RTKs with
phospho-MET the most significantly decreased (Fig. 4A). Quantification
of the phospho-RTK array showed that 15e decreased phospho-MET to
(
Fig. 2E).
1
/6 of that in the control group (Fig. 4B).
The above data suggest that tyrosine kinase MET could be the direct
3
.3. 15e inhibits Caki-1 cell migration and invasion in vitro
target for 15e, since MET is auto-phosphorylated [8]. If this hypothesis
is true, cells with high MET expression should be more affected by 15e.
To test this idea, seven different cancer cell lines with different levels of
MET and phosphorylated MET (Y1234/Y1235) were selected and tested
for their sensitivity to 15e. Caki-1, lung cancer cell NCI-H441 and gastric
carcinoma cell SNU-5 expressed high levels of total MET and phos-
phorylated MET (Y1234/Y1235), while breast cancer cell MCF7 and
MDA-MB-231, small cell lung cancer cell NCI-H446 and melanoma cell
SK-MEL-28 had low MET and phosphorylated MET expression (Fig. 4C
and D). Indeed, though there is considerable variation, Caki-1, NCI-
H441 and SNU-5 cells are very sensitive to 15e with IC50 0.60, 0.45 and
Given the above results, we further tested whether 15e can affect
Caki-1 cell migration and invasion in a wound healing assay and a trans-
well chamber assay, respectively. 15e at 0.625 and 5 M effectively
μ
blocked wound closure of Caki-1 cells (Fig. 3A). Similarly, in trans-well
chamber assay, treatment with 15e significantly decreased the number
of Caki-1 cells invading to the trans surface of trans-well chambers
(
Fig. 3B). In both assays, however, APG had minimum effect, again
demonstrating the improvement of 15e.
3
.4. Anti-proliferation activity of 15e correlates to phosphorylated MET
3
.92
μM, respectively. In stark contrast, the other cells were all less
responsive to 15e treatment with IC50 exceeding 10
μM (Fig. 4E and F).
Previously, APG was thought to have inhibitory activity against
Fig. 3. 15e inhibits Caki-1 cell migration and invasion in vitro. (A) Wound healing assays of Caki-1 cells treated with vehicle, 0.625
μ
M 15e, 5
μ
M 15e, 5
μ
M APG
or 5 M Crizotinib. (B) Trans-well chamber assays of Caki-1 cells treated by vehicle, 5 M 15e, 5 M APG or 5 M Crizotinib along with or without 15 ng/mL HGF.
μ
μ
μ
μ
Data are expressed as the mean ± SD, n = 3, *P < 0.05 versus Control. Not significant (n.s.) indicated P > 0.05.
7

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
Fig. 4. 15e induced anti-proliferation correlates to phosphorylated MET. (A) Phospho-RTK arrays of Caki-1 cells treated with vehicle or 5 M 15e for 24 hrs.
μ
Several phosphorylated RTKs were marked with black framework and number as indicated. (B) Quantification of 14 detectable Phospho-RTK expression in the assay
of phospho-RTK arrays. (C) Protein level of total MET and phosphorylated MET, and (D) quantification of relative p-MET/GAPDH protein level in seven cancer cell
lines. (E) Relative cell viability of seven cancer cell lines treated with 0.01–90
si-NC transfected Caki-1 cells. (H) Relative viability of si-MET or si-NC transfected Caki-1 cells treated with 15e. Data are expressed as the mean ± SD, n = 3, *P <
.05 versus si-NC.
μM 15e for 72 hrs and (F) their IC50 statistics (n = 3). (G) MET expression in si-MET or
0
8

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
The variation of IC50 in Caki-1, NCI-H441 and SNU-5 cells could be
contributed by the growth conditions of the different cancer cells.
To further confirm that it is the MET dependent cell growth of Caki-1
that was inhibited by 15e, we thought that knock down MET in Caki-1
cells with siRNA will decrease the inhibition by 15e. To that end, we
knocked down MET (Fig. 4G) and examined the anti-proliferation ac-
tivity of 15e. The results in Fig. 4H showed that this is the case, as si-
MET treated cells were less susceptible to 15e than control si-NC
treated Caki-1 cells. The data demonstrated that 15e was targeting
MET and inhibiting MET for inhibition of Caki-1 cell proliferation.
We further investigated how 15e affect the Akt kinase substrates
PRAS40 [46] and downstream pathway mTOR-p70-S6K [46]. In Fig. 6E
and F, phosphorylation of PRAS40, mTOR and p70-S6K were decreased
in Caki-1 cells treated with 15e in a dose-dependent manner.
3.7. Effects of 15e on mutant MET
Mutations in MET that activate its activity are found in hereditary
and sporadic papillary RCC. These include L1213V, V1238I, D1246N,
L1213V/D1246N/Y1248H
Y1248H, or M1268T [9,10]. Among these, MET
are
V1238I
resistant to Crizotinib [14] and Capmatinib [13,14], and MET
is
3
.5. 15e targets MET and inhibits its kinase activity
resistant to Cabozantinib [15]. To investigate whether these mutant
MET were also resistant to 15e, we transfected mouse embryonic
fibroblast NIH-3T3 cells with WT or mutant MET expressing plasmids
and incubated with 15e before detecting phosphorylation of MET. First,
WT and mutant MET were expressed at similar levels. As expected, the
mutant Met has higher kinase activity as shown by the higher levels of
phosphorylated MET at Y1234/Y1235 (Fig. 7A). As shown by the
immunoblot and quantification (Fig. 7B and C), 15e was able to inhibit
The decrease in phospho-MET seen in 15e treated Caki-1 cells
(
Fig. 4A) could be due to decrease in MET protein or MET phosphory-
lation. To clarify this point, we conducted immunoblot to examine MET
and Phospho-MET in 15e treated Caki-1 and NCI-H441 cells directly.
Phosphorylated MET (Y1234/Y1235), but not total MET, decreased with
increasing concentrations of 15e indicating that 15e inhibited MET
phosphorylation. As expected, the less active compound APG did not
affect MET phosphorylation at the same concentration (Fig. 5A and B).
Since Y1234/Y1235 are auto-phosphorylated [7], 15e was assumed
to inhibit the MET auto-phosphorylation kinase activity. To further
confirm this notion, we developed a Ba/F3 cell line, a mouse pro-B cell,
whose growth is dependent on the presence of IL-3 [42], by introducing
a translocated promoter region (TPR)-Met fusion gene to make the growth
of the cell dependent on MET in the absence of IL-3 [43]. The TPR-MET
contains a dimerization domain from TPR and a kinase domain from
MET, so that TPR-MET is constitutively active because of the auto-
dimerization [44]. In the Ba/F3 cells, inhibiting TPR-MET kinase ac-
tivity will lead to the suppression of cell viability. As expected, 15e was
found to only potently inhibit Ba/F3-TPR-MET cells with an IC50 0.44
V1238I
Y1248H
M1268T
the kinase activity of MET
, MET
and MET
as well as
MET . METD1246N, and METL1213V were also partially inhibited by 15e.
These data are significant as 15e could potentially be further developed
to treat Crizotinib, Capmatinib or Cabozantinib resistant patients who
WT
Y1248H
V1238I
H1112L
carry MET
resistant to 15e.
or MET
mutations. MET
, however, was
3.8. 15e inhibits tumor growth and MET phosphorylation in vivo
To examine the antitumor activity of 15e, we conducted a xenograft
tumor model in mice. Nude mice subcutaneously engrafted with Caki-1
cells were randomized and received intraperitoneal administration of
vehicle, 15e (20 mg/kg) or APG (20 mg/kg) for 16 days. At the termi-
nation of the experiment, tumor weight and body weight of the mice
were measured, and tumors were photographed (Fig. 8A). The mean
tumor weight of the vehicle control, 15e and APG groups were 1.35 g,
0.52 g and 1.40 g, respectively, and the difference between the 15e and
control group was statistically significant (p < 0.05) (Fig. 8B). There was
no difference in body weight among the groups (Fig. 8C). These data
demonstrate that 15e can effectively inhibit the tumor growth of renal
cancer cells in vivo.
μM (Fig. 5C). The activity of 15e was four times less in Ba/F3 cells in the
presence of IL-3 (IC50 = 1.69 M). The original APG has much less ac-
tivity in either cells (7.93 M and 15.38 M, respectively) (Fig. 5C). As
μ
μ
μ
expected, the MET phosphorylation in Ba/F3-TPR-MET cells were
significantly inhibited by 15e but not APG in a dose-dependent manner
with western blot analysis (Fig. 5D, with quantitation in Fig. 5E). The
results in this system further demonstrated that the target of 15e is
indeed MET.
Molecular docking of 15e to the available crystal structure of MET
kinase domain (PDB code 3F66) [45] further supported the above
conclusion. Docking of 15e (Fig. 5F) showed that 15e can fit into the
MET kinase pocket with the 5- hydroxyl- chromone group formed two
hydrogen bonds with the kinase hinge region residue M1160. Moreover,
the 5- hydroxyl- chromone group might also have hydrophobic in-
teractions with side chains of residues I1084, Y1159 and M1211. The 4-
hydroxy phenyl group could bind in the hydrophobic pocket surrounded
by side chains of residues V1092, L1140, L1157 and Y1230 and has
hydrogen bond with the hydroxy group of residue Y1230. The 4-acetyl
phenyl group fits in the lipophilic pocket surrounded by residues
M1211, Y1230 and D1231 (Fig. 5F).
The level of MET and phosphorylated MET in these tumor tissues
were further assessed. Immunohistochemical staining of MET and p-
MET-Y1234/Y1235 showed that while the total MET protein was not
significantly decreased in the 15e and APG treated mice, the phos-
phorylated MET at Y1234/Y1235 was visibly decreased in 15e treated
tumor tissues (Fig. 8D) while the decrease by APG is not obvious. These
data are consistent with the in vitro data in cell lines.
4. Discussion
Despite nephrectomy with curative intent, about one third of pa-
tients with clear cell RCC develop metastases which are associated with
high mortality [1]. Targeted therapies for RCC against VEGF and mTOR
have been developed, but treatment response is varied and most patients
eventually experience disease progression [47]. Therefore, there is still a
tremendous unmet medical need for RCC. Although many studies have
verified the anti-tumor activity of APG in different cancer cell lines, the
activity of APG is usually too low to be further developed [22]. However,
some studies reported that structural modification of APG can elevate its
anti-proliferation activity in multiple cancer cell lines [27,28], and
especially a APG derivative exhibited strong activity against colorectal
adenocarcinoma (HT-29) and leucocythemia (HL-60) cells with IC50
value of 2.03 µM and 2.25 µM, respectively, which were better than 5-
fluorouracil [28]. Here we synthesized a series of novel APG de-
rivatives with difference at the Ar group (Scheme 2) and found that a
derivative 15e, with a para-acetylphenyl nonpolar group, had potent
3
.6. 15e downregulates MET downstream Akt and Stat3 signaling
As a receptor tyrosine kinase, MET was the upstream regulator of
several classical downstream pathways including PI3K-Akt, Ras-Raf-
MEK-Erk and Stat3 [8]. The effect of 15e on the downstream three
pathways were investigated in Caki-1 cells. Indeed, phosphorylated Akt
(
S473 or T308) and phosphorylated Stat3 (Y705), but not phosphory-
lated Erk1/2 (T202/Y204) were downregulated by 15e in a dose-
dependent manner (Fig. 6A and B). As expected when HGF was added
to activate MET, Akt and Stat3 phosphorylation were increased and 15e
can still inhibit this HGF-induced enhancement of Akt and Stat3 phos-
phorylation (Fig. 6C and D). It indicated that 15e can block the signaling
from MET to Akt and Stat3.
9

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
Fig. 5. 15e directly targets MET kinase domain and inhibits its kinase activity. (A) Immunoblot of MET and p-MET-Y1234/Y1235 in Caki-1 and NCI-H441 cells
treated with 0 and 1.25–20 M 15e or APG for 16 hrs. (B) Quantification of relative p-MET/MET protein level in (A). (C) Relative viability of Ba/F3-WT and Ba/F3-
μ
TPR-MET cells treated with concentration gradients of 15e or APG for 72 hrs. (D) Immunoblot and (E) its analysis of TPR-MET phosphorylation in Ba/F3-TPR-MET
cells treated with indicated concentrations of 15e or APG. (F) 15e docked into the ATP-binding cleft of MET. 15e is shown as yellow ball and stick. The key amino
acid residues are shown as green stick. Hydrogen bonds are shown as magenta dashed lines. Data are expressed as the mean ± SD, n = 3, *P < 0.05 versus Control (0
μ
M) in (B) and (E), or *P < 0.05 versus group of Ba/F3 + IL3 in (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
1
0

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
Fig. 6. 15e downregulates MET downstream Akt and Stat3 signaling. (A) Immunoblot analysis and (B) its quantification of MET downstream protein in Caki-1
cells treated with 0 and 1.25–20 M 15e for 16 hrs. (C) Immunoblot analysis and (D) its quantification of MET downstream protein in HGF stimulated Caki-1 cells
treated with 0 and 1.25–20 M 15e. The cells were pre-incubated by 15e for 3 hrs before 50 ng/ml HGF stimulation for 15 min. (E) Immunoblot analysis and (F) its
quantification of Akt downstream protein in Caki-1 cells treated with 0 and 1.25–20 M 15e for 16 hrs. Data are expressed as the mean ± SD, n = 3, *P < 0.05 versus
M 15e without HGF and *P < 0.05 versus group of 0 M 15e with HGF in (D).
μ
μ
μ
#
group of 0
μ
M 15e in (B) and (F). P < 0.05 versus group of 0
μ
μ
activity inhibiting Caki-1 cell proliferation (IC50 = 0.49
fold improvement than that of APG (Table 1). Furthermore, the activity
of 15e is 10 times better than that of Crizotinib (IC50 = 4.43 M,
Table 1), and is also better than the reported IC50 (IC50 = 14.5 M in
μ
M) with a 90-
Caki-1 cell) for the approved RCC drug Cabozantinib [16]. Therefore, we
discovered a new molecular entity 15e with effective anti-RCC activity
via structural modification of APG.
μ
μ
Identification of the precise target of APG or its derivative has been a
1
1

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
Fig. 7. Effects of 15e on phosphorylated activation of mutant MET. (A) MET and p-MET-Y1234/Y1235 in NIH-3 T3 cells transfected with WT or mutant MET
plasmid. (B) p-MET-Y1234/Y1235 of WT or mutant MET in NIH-3 T3 cells treated by 0 and 0.156–10 M 15e for 24hrs. (C) Quantification of relative p-MET/MET
level of WT or mutant MET under the treatment of 15e concentration gradients. Data are expressed as the mean ± SD, n = 3.
μ
challenge. Natural flavonoid APG was discovered several decades ago
48], and many mechanism investigations have been performed.
inhibitor.
MET has been shown to play critical role in a number of cancers
[
Although APG was demonstrated to affect the expression of many pro-
teins and multiple signal transduction pathways [22], the precise target
of APG has not been conclusively identified. A comprehensive study
using phage display with high-throughput sequencing found more than
including RCC [4]. Gene amplification and mutations in MET activates
downstream pathways and lead to tumor cell proliferation, tumor in-
vasion and pathogenesis [4]. Therefore, if 15e targets MET, it should
affect the MET downstream pathways. Our data confirmed that of the
three MET classical downstream pathways, PI3K-Akt, Ras-Raf-MEK-Erk
and Stat3, 15e mainly affected activation of Akt and Stat3 rather than
Erk1/2 in Caki-1 cells stimulated with or without HGF (Fig. 6).
Consistent with these data, cells that have high MET and are more
dependent on MET for growth are more sensitive to 15e (Fig. 4C–H).
Furthermore, with the artificial system where Ba/F3 cells was made to
depend on MET, inhibition of TPR-MET kinase activity led to the sup-
pression of cell viability while the activity of 15e was four times less in
Ba/F3 cells in the presence of IL-3 (Fig. 5C). Collectively, our data
indicate that 15e directly targeted MET, and inhibited MET auto-
phosphorylation and its downstream pathways for its anti-RCC activity.
Overcoming MET mutation, especially drug resistant mutation, is
important for clinical application. In hereditary and sporadic papillary
RCC, MET has been found carrying many constitutively activated mu-
tation such as H1112L, L1213V, V1238I, D1246N, Y1248H, or M1268T
1
60 APG-binding proteins [23], but also missed some target proteins
from other studies [24–26]. The difficulty is partly due to the low po-
tency of APG. Improving the potency is challenging because some of the
derivative compounds aggregate at high concentrations and because of
their natural fluorescence/color and hence there are arguments whether
they were pan-assay interference compounds (PAINS) [49,50]. Now
with the more potent APG derivative 15e, we provided a good tool
compound to identify the true target for APG. The kinase panel data
(
Fig. 4) clearly showed that 15e was specifically inhibiting phosphory-
lation of MET but not other RTKs such as EGFR and RET, and the activity
and selectivity were further supported by results shown in Fig. 4 that
1
5e selectively repressed proliferation of cells with high MET phos-
phorylation. 15e specifically inhibited RCC in functional experiments in
vitro and vivo (Fig. 2, 8A). And since MET undergoes auto-
phosphorylation, we conclude that MET is the direct target of 15e.
Docking modeling of 15e into MET kinase domain fit this hypothesis and
the modification at the Ar group indeed make sense (Fig. 5F). These
multiple results all supported that 15e is an improved selective MET
L1213V/D1246N/Y1248H
L1213V/D1246N/Y1248H
[9–11], while MET
, MET
were resistant to approved MET inhibitor Crizotinib, Cap-
matinib and Cabozantinib, respectively [12–15]. When tested against
and
V1238I
MET
1
2

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
Fig. 8. 15e inhibits tumor growth and MET phosphorylation in vivo. (A) Photograph of tumors from vehicle, 15e and APG treated mice. (B) Weight of Caki-1
xenograft tumors from nude mice 16 days after treatment. (C) Body weight of mice after treatment. (D) Immunohistochemical staining of Met and p-MET-Y1234/
Y1235 in Caki-1 xenograft tumor tissues. Data are expressed as the mean ± SD, n = 6, *P < 0.05 versus Vehicle.
these mutations, 15e was found to still have activity against METV1238I,
resistant mutant METV1238I/Y1248H can also be abrogated by 15e. In vivo
study confirmed the inhibitive activity of 15e in RCC growth and MET
phosphorylation. These results indicate 15e is a promising novel
Y1248H
M1268T
WT
D1246N
MET
MET
and MET
as well as MET , while MET
and
L1213V
were partially inhibited (Fig. 7). Neither of V1238, Y1248
and M1268 in MET kinase domain was key interaction sites of 15e
candidate for RCC therapy, and especially provide a potential alterna-
V1238I/Y1248H
(
Fig. 5F), which may explain why 15e still showed activity against
tive to overcome drug-resistant MET
in RCC.
V1238I
Y1248H
M1268T
MET
, MET
and MET
. However, D1246, L1213 and
especially H1112 were likely important sites for interaction with 15e at
MET kianse domain. Collectively, these data are important as 15e could
be further developed to treat Crizotinib, Capmatinib or Cabozantinib
CRediT authorship contribution statement
Jing Li: Conceptualization, Methodology, Validation, Formal anal-
ysis, Investigation, Writing - original draft, Project administration.
Guishan Tan: Supervision, Funding acquisition. Yabo Cai: Conceptu-
alization, Methodology, Validation, Formal analysis, Investigation,
Writing - original draft. Ruihuan Liu: Supervision. Xiaolin Xiong:
Investigation. Baohua Gu: Validation, Formal analysis, Writing - orig-
inal draft. Wei He: Investigation. Bing Liu: . Qingyun Ren: Concep-
tualization. Jianping Wu: Validation. Bo Chi: Writing - original draft.
Hang Zhang: Writing - original draft. Yanzhong Zhao: Validation,
Formal analysis. Yangrui Xu: Investigation. Zhenxing Zou: Validation,
Formal analysis. Fenghua Kang: Validation, Formal analysis. Kangping
Xu: Conceptualization, Supervision, Funding acquisition.
Y1248H
V1238I
resistant patients who carry MET
or MET
mutations. The
activity against the mutant MET implies that the interaction site of 15e
with MET is different from previous compounds and indicate that there
are potential novel MET inhibitors to be further discovered.
In summary, a series of APG derivatives were synthesized and
screened for their anti-RCC activity on Caki-1 cell in vitro, and compound
1
5e was the most potent. 15e was further shown to be able to inhibit cell
colony formation, neoplasm growth in 3-D microsphere assay, migration
and invasion. Mechanistically, 15e caused cell cycle arrest but not
apoptosis. At the molecular level, 15e inhibit auto-phosphorylation of
MET at Y1234/Y1235 likely by directly interact with MET kinase
domain. As expected, HGF-MET downstream pathways, Akt and Stat3
WT
were downregulated by 15e. Importantly, in addition to MET , drug
1
3

J. Li et al.
Biochemical Pharmacology 190 (2021) 114620
Declaration of Competing Interest
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