Structure-activity relationship of spop inhibitors against kidney cancer

Article abstract of DOI:10.1021/acs.jmedchem.0c00161

Speckle-type POZ protein (SPOP) is overexpressed in the nucleus and misallocated in the cytoplasm in almost all the clear-cell renal cell carcinomas (ccRCCs), which leads to kidney tumorigenesis. Previously, we elucidated that the oncogenic SPOP-signaling pathway in ccRCC could be suppressed by 6b that inhibits SPOP-mediated protein interactions. Herein, we have established a structure-activity relationship for 6b analogues as SPOP inhibitors. Compound 6lc suppresses the viability and inhibits the colony formation of ccRCC cell lines driven by cytoplasmic SPOP, superior to 6b. Compound 6lc binds to the SPOP protein in vitro and disrupts SPOP binding to phosphatase-and-tensin homologue (PTEN) in HEK293T cells, which causes the observable phenomena: a decline in the ubiquitination of PTEN, elevated levels of both PTEN and dual-specificity phosphatase 7, and decreased levels of phosphorylated AKT and ERK when ccRCC cell lines are exposed to 6lc in a dose-response manner. Taken together, compound 6lc is a potent candidate against kidney tumorigenesis.

Full text of DOI:10.1021/acs.jmedchem.0c00161

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Article
Structure-activity Relationship of SPOP Inhibitors Against Kidney Cancer
Ze Dong, Zhen Wang, Zhong-Qiang Guo, Shouze Gong, Tao
Zhang, Jiang Liu, Cheng Luo, Hualiang Jiang, and Cai-Guang Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00161 • Publication Date (Web): 16 Apr 2020
Downloaded from pubs.acs.org on April 17, 2020
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Structure-activity Relationship of SPOP Inhibitors Against
Kidney Cancer
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†,§
¶,†,§
Zhong-Qiang Guo,,# Shouzhe Gong, Tao Zhang, Jiang
†
†
Ze Dong, Zhen Wang,
#
†,‡
†,‡,
Cai-Guang Yang†,‡,*
Liu, Cheng Luo, Hualiang Jiang,
†
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China
¶
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023,
China

Department of Urology, Zhongnan Hospital of Wuhan University, Hubei 430071,
China
#
CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of
Genomics, Chinese Academy of Sciences, Beijing 100101, China
‡
School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced
Study, UCAS, Hangzhou 310024, China

Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTec University,
Shanghai 201210, China
Keywords:
cytoplasmic SPOP, inhibitor, protein-protein interaction, ccRCC, kidney cancer
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ABSTRACT
Speckle-type POZ protein (SPOP) is overexpressed in the nucleus and misallocated in
the cytoplasm in almost all the clear-cell renal cell carcinomas (ccRCC), which leads
to kidney tumorigenesis. Previously, we elucidated that the oncogenic SPOP-
signaling pathway in ccRCC could be suppressed by 6b that inhibits SPOP-mediated
protein interactions. Herein, we have established a structure-activity relationship for
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b analogs as SPOP inhibitors. Compound 6lc suppresses the viability and inhibits the
colony formation of ccRCC cell lines driven by cytoplasmic SPOP, superior to 6b.
Compound 6lc binds to SPOP protein in vitro, and disrupts SPOP binding to PTEN in
HEK293T cells, which causes the observable phenomena: a declined in the
ubiquitination of PTEN, elevated levels of both PTEN and DUSP7, and decreased
levels of phosphorylated AKT and ERK when ccRCC cell lines are exposed to 6lc in a
dose-response manner. Taken together, compound 6lc is a potent candidate against
kidney tumorigenesis.
INTRODUCTION
Kidney cancer is resistant to chemotherapy in clinic and usually metastasizes once
the disease has already been diagnosed. Several drugs have been approved for the
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targeted therapy of kidney cancers. Typically, these drugs are either kinase
inhibitors for vascular endothelial growth factor (VEGF) and platelet-derived growth
factor (PDGF) receptors or inhibitors for the mammalian target of rapamycin
(mTOR). Significantly, the first checkpoint inhibitor Nivolumab, a monoclonal
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antibody targeting PD-1, was approved in clinic for renal cell carcinomas (RCC)
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therapy. Bevacizumab, another monoclonal antibody targeting the VEGF receptor,
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also shows good antitumor activity in patients with metastatic RCC. Recently, the
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small-molecule inhibitor PT2399 was shown to successfully disrupt the HIF 2α-Arant
interaction and to suppress the progression of clear-cell RCC (ccRCC). PT2385, the
analog of PT2399, has been approved for the clinical research.4, 5 Although these
anti-tumor drugs and candidates are available for the treatment of ccRCC, the dual
challenges of low response rate and drugs resistance demand urgent exploration of
untargeted targets and underexplored chemical spaces for targeted therapy of
ccRCC.
The nuclear speckle-type POZ protein (SPOP) plays important roles within the
6-8
ubiquitination machinery in cancers. SPOP contains an N-terminal MATH domain
(SPOPMATH) for selectively binding to a substrate protein and a C-terminal BTB
adaptor domain for E3 ubiquitin ligase recognition.9-11 Remarkably, the E3 ligase
adapter SPOP is overexpressed in 85% of kidney cancers; accumulation of dislocated
SPOP in the cytoplasm occurs in nearly 100% of the two predominant forms of
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kidney cancer, namely primary and metastatic ccRCC. The cytoplasmic SPOP serves
as a regulatory hub in order to promote cancer through the ubiquitination and
degradation of multiple cancer proteins that are involved in cellular proliferation and
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apoptosis. The two tumor suppressors, phosphatase-and-tensin homolog (PTEN)
and dual-specificity phosphatase 7 (DUSP7) are central to the mechanism in the
oncogenic SPOP-signaling. Binding of SPOP to PTEN and DUSP7 in the cytoplasm
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degrades these two proteins, which inactivates its downstream AKT/ERK signaling
pathways, and which eventually promotes ccRCC tumorigenesis. Indeed, the
biological rationale is compelling, since the inhibition of SPOP-mediated protein
interactions and the stabilization of PTEN and DUSP7 proteins would be beneficial
for the treatment of ccRCC.
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We have established that small-molecule targeting of the E3 ligase adaptor SPOP
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could be a specific approach against ccRCC. Compounds 6a and 6b block the
interaction of the SPOP ubiquitin ligase with its substrates (Figure 1), thus leading to
the impaired proliferation of ccRCC cell lines that characteristically have cytoplasmic
SPOP. Our discovery of compound 6b convincingly demonstrates on a biochemical
level in cells the therapeutic potential of blocking SPOP activity to stabilize PTEN and
DUSP7 in order to suppress the AKT- and ERK-driven cancer-signaling pathways,
which serves as “proof-of-principle” in order to manipulate the “undruggable” E3
ligase in the ubiquitin-proteasome system in ccRCC.15, 16
Figure 1. Structures of our previously reported SPOP inhibitors. Compound 6c is a
negative control.
In this study, we explored more chemical spaces and attempted to establish a
structure-activity relationship (SAR) in the aspect of medicinal chemistry for SPOP
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inhibitors. Several analogs of 6b are shown to suppress the proliferation of ccRCC cell
lines typically with single-digital micro-molar IC values. Among them, compound 6lc
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is active against kidney cancer cell lines through the inhibition of the SPOP-mediated
ubiquitination-signaling pathway in ccRCC.
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RESULTS AND DISCUSSION
Chemical Synthesis. We performed structural optimization based on chemical
scaffold of the established SPOP inhibitors in several aspects using medicinal
chemistry strategy,17, 18 including 1) N-substituents, 2) various amide side-chains, 3)
the impact of methyl group position, 4) carboxylic acids and esters, and 5)
conjugation shrinking (Figure 2).
Figure 2. Design of different sets of SPOP inhibitors.
First, we investigated the effects of various N-substituents attached to cyclic
amidine. Scheme 1 shows the synthetic route for compounds 6d-6z. Condensation of
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2
-methylpyridin-2-amine 1 and diethyl malonate at elevated temperature provides
-hydroxy-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one 2. Simultaneous chlorination
and formylation of 2 give the intermediate 3 under Vilsmeier-Haack reaction. Various
imines 4 are obtained in good yields by treating the intermediate 3 with
corresponding amines in the presence of anhydrous Na SO in EtOH at room
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temperature (rt). Lastly, refluxing imines 4 with α-cyanoacetamide 5a in CHCl3
provides a series of target compounds 6d-6z in high yields.
Scheme 1. Synthesis of compounds 6d-6za
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R1
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Compound
d/6d
Compound
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4l/6l
4t/6t
4e/6e
f/6f
4g/6g
h/6h
4m/6m
4n/6n
4o/6o
4p/6p
4u/6u
4v/6v
4w/6w
4x/6x
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4i/6i
4q/6q
4y/6y
4z/6z
4
j/6j
4r/6r
4s/6s
4
k/6k
a
o
Reagents and conditions: (a) 220 C, diethyl malonate, neat; (b) POCl , DMF; (c)
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R NH , Na SO , EtOH, rt, overnight; (d) 5a, piperidine, CHCl , reflux, overnight; (e)
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neat, rt, 3 days.
Next, we introduced various amide sidechains into the main scaffold where the R1
group is held constant as thiophenylethyl, because the sulfur-containing heterocycle
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substituent might make the target compounds more “drug-like” and soluble. When
α-cyanoacetamides 5b-5k are employed as reagents in the one-step condensation
with intermediate 4, compounds 6la-6lj are obtained in high yields (Scheme 2).
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Scheme 2. Synthesis of compounds 6la-6lja
R2
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Compound
b/6la
Compound
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5g/6lf
5c/6lb
d/6lc
5e/6ld
f/6le
5h/6lg
5i/6lh
5j/6li
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5k/6lj
a
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Reagents and conditions: (a) R NH , neat, rt, 3 days; (b) piperidine, CHCl , reflux,
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overnight.
To investigate the impact of the methyl group attached to the planar tricycle (Figure
3), we synthesized 9-methyl substituted compounds 6lk and 6ll without methylation
in a similar route for the synthesis of compounds 6la-6lj.
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Figure 3. Compounds with or without methyl group.
Furthermore, we prepared a set of esters 7a-7j and the corresponding carboxylic
acids 8a-8j (Scheme 3). Ethyl esters 7 are synthesized in high yields through the
reactions between compound 4 and ethyl α-cyanoacetate 5l. Compounds 8a-8j are
obtained from alkaline hydrolysis of the corresponding ethyl esters 7a-7j.
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Scheme 3. Synthesis of compounds 7 and 8a
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overnight; (b) LiOH, THF/EtOH/H O, 45 C, overnight.
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Lastly, we synthesized several bicyclic compounds in order to investigate the effect
of polycyclic conjugation on the inhibitory activity of SPOP inhibitors. The synthetic
route for the preparation of compounds 13 and 14 is depicted in Scheme 4.19, 20
Compound 9 is reacted with diethyl malonate to give ethyl ester 10. Alkylation of 10
with (2-bromoethyl)benzene affords N-alkylated lactam 11. Alkaline hydrolysis of 11
followed by condensation of carboxylic acids and amines provide target compound
carboxamides 13 and 14 in high yields.
Scheme 4. Synthesis of compounds 13 and 14a
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DIEA, DMF.
Nano Differential Scanning Fluorimetry (nanoDSF) screening for SPOP binders. The
nanoDSF assay that measures the thermal unfolding transitions (Tm) of an interested
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protein under native conditions, no extra dye is required. In order to evaluate
whether our newly synthesized compounds bind to SPOP protein in vitro, we
performed a high-throughput screening for the Tm shift (ΔTm) of SPOPMATH induced
by compounds (Table 1 and S1). Several compounds could stabilize SPOPMATH protein
o
with ΔTm above 3.0 C when the 10-fold compound was subjected to the nanoDSF
assay, while compound 6c, the negative control had minimal effect on the thermal
stability of SPOPMATH. To our delight, the ΔTm could be further shifted up to 4.0 oC
when the 20-fold compound 6b was assayed. In addition to 6b, compound 6e, 6g-i,
6
l, 6lc, 6ld, 7f, and 8c as listed in Table 1 stabilize SPOPMATH as showing ΔTm values
o
over 3.5 C, alongside compounds 6r, 6lf, 6lj, 7c, and 8b as shown in Table S1. Since
the SPOP binder 6b has been shown to be active against kidney cancer through
targeting the SPOP-mediated signaling pathway in ccRCC, the nanoDSF assay could
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be the first step of screening for small-molecule binders of SPOP that may be
potential to inhibit SPOP-mediated protein interactions in the further biological
annotation.
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Table 1. Tm shift of SPOPMATH in the presence of compoundsa
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ΔTm ( C)
0 
-1.7
2.8
0
1.8
2.5
2.5
2.4
2.3
3.7
3.2
ΔTm ( C)
Compound
Compound
1
20 
-3.8
4.0
0.2
2.1
3.6
2.9
3.5
3.5
5.3
5.1
10 
20 
DMSOb
6b
6la
6lb
6lc
6ld
6li
7d
7e
7f
2.8
3.1
3.1
3.5
2.7
2.6
3.3
3.5
3.7
2.7
6
c
6
6
d
e
6
f
0.8
0.8
4.9
1.7
2.3
1.6
1.5
5.3
1.9
3.7
6
g
6
h
6i
7j
8c
6
l
a
Data was calculated using DMSO-treated SPOPMATH as the reference. Data was
b
obtained using naive SPOPMATH sample as the reference.
Antiproliferation of SPOP binders in ccRCC cell lines. Next, we performed an MTT
assay in order to evaluate the antiproliferative effect of those SPOP binders
identified in the nanoDSF screening on kidney cancer cells. The two ccRCC cell lines
A498 and OS-RC-2 were assayed (Table S2). The known SPOP inhibitor 6b was
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assayed as the positive control, which inhibits the proliferation of A498 and OS-RC-
2 cells up to 88% and 90% at 30 μM, respectively. Compound 6c, the negative control
minimally attenuates the proliferation of the two ccRCC cell lines at high
concentrations. Several compounds, for example, 6d-6i, 6k-6l, 6n, 6lc, and 6li,
exhibited comparable antiproliferative activities with compound 6b. Of note, those
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compounds are SPOP binders too, which suggests that the nanoDSF is a validated
assay for the initial screening of SPOP inhibitors.
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2
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2
2
2
2
2
2
2
2
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6
0
1
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6
7
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9
0
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9
0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Analysis of SARs. With these newly designed 6b analogs in hand, we sought to
analyze the SAR as of SPOP inhibitors. First, we investigated the effects of the R1
substitutes on the inhibition of cell proliferation (Table 2). Introduction of methyl
(6d), methoxyl (6e), fluoro (6f), chloro (6g), and bromo (6h) substitutes on the para-
position of the phenethyl motif has negligible impacts on inhibitory activities, which
display antiproliferative rates ranging between 88% and 97% against A498 and OS-
RC-2 cells at 30 μM, comparable to compound 6b. Interestingly, changing the
position of fluoro group from the para-position (6f) or the meta-position (6i) to the
ortho-position (6j) is unfavorable to the cell inhibitory activities, since the inhibitory
rates of 6j are decreased in both ccRCC cell lines. 2-(diethylamino)ethyl (6k) bearing
an acyclic apolar substitute exhibits good cellular activity, with inhibitory rates of
between 86% and 89% against the proliferation of A498 and OS-RC-2 cells,
respectively. Compared to the thiophenylethyl-substituted compound 6l, 2-
morpholinoethyl-substituted analog 6m exhibits declined inhibitory potency. 3-
phenylpropyl-substituted compound 6n largely suppressed cell proliferation, while
an introduction of an oxygen atom into the chain of compound 6n causes 2-
phenoxyethyl-substituted compound 6o to dramatically lose activity. Compound 6p
bearing 3-morpholinopropyl exhibits declined antiproliferative activities against both
A498 and OS-RC-2. Compound 6q with benzyl substituent exhibits declined activities,
with 58% and 46% inhibitory rate against the proliferation of A498 and OS-RC-2 cells.
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A similar phenomenon is observed on compound 6r that having pyridin-4-ylmethyl
group replaced benzyl substituent. Compounds 6s and 6z bearing aromatic groups
exhibit attenuated antiproliferative activities against the two ccRCC cell lines, with
inhibitory rates of 12% and 17% in A498 cells and 23% and 38% in OS-RC-2 cells,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
respectively. Compounds (6t, 6u, 6v, and 6w) with the cyclic or linear R -substituted
alkyl group also showed reduced inhibitory activity compared to 6b. Lastly, the
introduction of propargyl (6x) or the bulky indole (6y) proves unfavorable to
antiproliferation. Taken together, results indicate that the introduction of polar
substituents into the nitrogen is generally unfavorable to overall inhibitory potency
in ccRCC cell lines.
Table 2. The SARs of N-substituents
Inhibition
@ 30 μM (%)
A498 OS-RC-2
Inhibition
@ 30 μM (%)
A498 OS-RC-2
Compound
Compound
6
6
6
6
6
b
d
e
f
g
88
91
90
90
90
89
89
61
86
90
35
90
90
94
88
88
97
96
92
74
89
82
40
91
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
6z
50
27
58
48
12
37
59
11
57
37
55
17
36
35
46
35
23
22
62
15
66
37
51
38
6
h
6i
6
6
6l
j
k
6
m
6
n
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Next, we found that variations of amide sidechains in SPOP inhibitors have
significant effects on the inhibition of proliferation in ccRCC cell lines (Table 3).
Changing the 3-morpholinopropyl (6l) to the 3-phenylpropyl (6la) substituent
reduces antiproliferative activities, with moderate inhibitory ratios of 62% and 58%
for compound 6la on A498 and OS-RC-2 cells at 30 μM, respectively. In general,
compound 6lb-ld, and 6lf bearing the short amide sidechains largely retained their
inhibitory activities against the proliferation of A498 cells. Surprisingly, compound
1
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1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
le, with 4-fluorophenethyl substituent, exhibits a significantly declined activity
against proliferation, with inhibitory ratios of 12% and 21% for A498 and OS-RC-2
cells at 30 μM. The benzyl-amide substituted compound 6lg also becomes less
active. Of note, compounds 6lh and 6li with cyclic substituents largely maintain
potency for inhibition, while the introduction of β-aminoalcohol (6lj) exerts only
slight influence on antiproliferative activity.
Table 3. The SARs of amide sidechains
Inhibition
Inhibition
Compound
@ 30 μM (%)
Compound
@ 30 μM (%)
A498
OS-RC-2
58
A498
OS-RC-2
6
la
6lb
lc
ld
6le
62
86
85
85
12
6lf
6lg
6lh
6li
87
60
86
90
83
75
50
83
90
81
82
90
52
21
6
6
6lj
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4
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5
5
5
6
Subsequently, we investigated the impact of the methyl substituent on the planar
tricycle. We compared the antiproliferative activities of compounds without the
methyl group or with the methyl on different positions (Table 4). Compound 6lk with
9-methyl substituent and 6ll without methyl group showed attenuated activities
against the proliferation compared with compound 6l with 10-methyl substituent,
thus indicating that the position of the methyl substituent in SPOP inhibitor is also
important for the antiproliferation in ccRCC cell lines.
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
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9
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1
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8
9
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Table 4. The impact of methyl group
Methyl
position
Inhibition @ 30 μM (%)
Compound
A498
90
OS-RC-2
82
1
0-Me
-Me
No-Me
6l
6lk
6ll
9
75
77
62
56
As depicted in Table 5, most esters show reduced activities against the proliferation
of A498 and OS-RC-2 cells compared to the corresponding amides (Table 2).
Compound 7g is the most active SPOP inhibitors among the 10 esters, but only
achieves less than 60% antiproliferation in the two ccRCC cell lines at 30 μM. In
addition, no carboxylic acids (8a-j), hydrolyzed from the corresponding esters (7a-j)
under alkaline condition, show satisfied activities against ccRCC cell lines. In general,
the esters or carboxylic acids as SPOP inhibitors are much less active than the amide
derivatives.
Table 5. The SARs of esters and carboxylic acids
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Inhibition
@ 30 μM (%)
A498 OS-RC-2
27 23
Inhibition
@ 30 μM (%)
1
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Compound
Compound
A498
18
OS-RC-2
22
7
a
8a
8b
8c
8d
8e
8f
8g
8h
8i
7b
38
24
42
51
38
59
24
51
33
35
20
48
49
49
56
26
10
16
21
13
57
51
25
24
20
24
18
29
28
30
31
37
16
32
16
33
7
c
7
7
d
e
7
f
7
g
7
h
7i
7
j
8j
Lastly, in order to diversify the chemical scaffold of SPOP inhibitor, we shrank the
tricyclic coplanar conjugation to the bicyclic system. We synthesized several bicyclic
compounds with different substituents, including acids, esters, and amides (Table 6).
Unfortunately, these bicyclic analogs usually display low antiproliferative activity in
the two ccRCC cell lines, thus suggesting the important contribution of tricyclic
coplanar conjugation on SPOP inhibitors.
Table 6. The SARs of planner conjugation
Inhibition
@ 30 μM (%)
Inhibition
@ 30 μM (%)
A498
Compound
X = N, Y = C
Compound
X = C, Y = N
R2
A498
OS-RC-2
OS-RC-2
11a
12a
15
33
19
24
11b
12b
14
21
15
15
16
1
3a
8
14
14a
25
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4
4
4
4
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5
5
5
5
6
13b
13
30
14b
38
11
Quantitation of antiproliferative activity of SPOP inhibitors. Following the SARs
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7
8
9
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9
0
analysis of SPOP inhibitors, we quantitatively determined the IC values for those
50
compounds displaying >75% antiproliferative activities in both ccRCC cell lines at a
single dose of 30 μM (Table 7). The IC of compound 6b is 2.7 μM and 5.8 μM
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against A498 and OS-RC-2 cell lines, respectively, which is similar to our previously
reported results. Compound 6c is assayed as a negative control. In general, most
tested compounds exhibited comparable activities to 6b for the inhibition of ccRCC
cell proliferation with single-digital micro-molar IC values, particularly those
5
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1
compounds containing apolar alkyl groups at the R -position. Compound 6d-6i with
phenethyl substitution, compound 6k bearing acyclic 2-(diethylamino)ethyl
1
sidechain, and compound 6l containing heterocyclic substituent at the R positions
show comparable IC values to inhibitor 6b. Similarly, compound 6n with 3-
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phenylpropyl displays comparable activity to 6b, while 6w bearing the propyl group
shows 2-fold drops in IC value for A498 cells. In line with the activities observed in
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single dosage MTT assay, the compounds bearing various amide sidechains also
display potent antiproliferation against the two ccRCC cell lines, including 6lc, 6lf,
and 6li. Compound 6ld is much less active against the proliferation in OS-RC-2 cells
with an unknown mechanism.
Table 7. The antiproliferative activity of selected inhibitors
IC (μM)
IC (μM)
50
5
0
Compound
Compound
A498
2.7  1.0
OS-RC-2
5.8  1.2
A498
3.5  0.7
OS-RC-2
3.9  0.8
6
b
6k
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c
> 50
> 50
6l
6n
6w
6lc
6ld
6lf
6li
2.6  0.5
2.6  0.2
6.4  0.9
2.1  0.7
5.0  1.3
2.8  0.7
6.3  0.5
7.6  2.1
5.1  0.8
5.2  0.6
3.5  0.5
24.2  2.8
4.2  0.7
5.9  0.8
6
d
2.1  0.6
2.8  0.4
3.1  0.8
2.3  0.3
3.0  0.5
2.4  0.4
5.7  1.2
6.5  1.1
6.6  1.0
4.0  0.8
5.0  1.0
4.6  0.6
6e
6
f
6
g
1
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0
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
6h
6
i
Investigation of antiproliferative selectivity of 6lc. Although SPOP localizes in the
nucleus and SPOP mutations are common in a variety of cancers, the ccRCC cell lines
are unique in that overexpression of SPOP leads to ectopic localization in the
cytoplasm. To this end, the SPOP inhibitor could selectively attenuate proliferation in
ccRCC cell lines, but not in cell lines in which SPOP is absent from the cytoplasm. As
expected, 6lc displays antiproliferation in the two ccRCC cell lines of A498 and OS-
RC-2 with IC of 2.1 μM and 3.5 μM, respectively (Figure 4A), while 6lc minimally
5
0
inhibits the growth of the non-tumor kidney cell line HK-2 (human proximal tubule
epithelial cell line). In our SAR exploration, compound 6lc is not sharply different
from 6h and 6lf and our previously reported 6b in potency against the proliferation
of ccRCC cell lines (Table 7). These inhibitors also had similar thermal effects on the
stabilization of SPOP protein in vitro (Table 1). However, the solubility of 6lc is
determined to be >90 μM in cell culture medium in the presence of 0.2% DMSO,
which is much more soluble than 6b (30-45 μM), 6h (5-10 μM), and 6lf (2.5-5 μM). In
addition, 6lc significantly inhibited the colony formation for both A498 and OS-RC-2
cells, superior to 6b, 6h, and 6lf (Figure 4B). Taken these data together, we chose 6lc
as the representative inhibitor of SPOP-protein interaction for further investigations.
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8
9
0
Figure 4. Antiproliferative effect of inhibitor 6lc. (A) Quantitation of IC of 6lc
5
0
against the proliferation of ccRCC cell lines and HK-2 cells. (B) Effect of SPOP
inhibitors on the colony formation of A498 and OS-RC-2 cell lines. (C) Effect of 6lc on
the proliferation in HEK293T cells overexpressing SPOP. (D) Effect of 6lc on cell
proliferation in other cancer cell lines. **, p < 0.01; ***, p < 0.001; unpaired
Student's t-test. Error bar, mean  SD, n = 3.
It has been previously shown that HEK293 cells do not express SPOP in the
cytoplasm, but the overexpression of SPOP in HEK293T cells promotes
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tumorigenesis. As expected, the recapitulated ccRCC phenotype observed in the
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HEK293T cells of overexpressing SPOP could be suppressed by the treatment of the
SPOP inhibitor 6lc (Figure 4C), thus indicating compound 6lc is specific to the
inhibition of the cytoplasmic SPOP. Lastly, we also determined the effect of 6lc on
proliferation in other cancer cell lines in which SPOP is absent from the cytoplasm.
By over five folds the IC for A498 cell proliferation measured less than that for four
1
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0
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0
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0
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9
0
1
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7
8
9
0
5
0
other tested cell lines (Figure 4D). Taken together, SPOP inhibitor 6lc is likely specific
to suppress the proliferation of cells driven by cytoplasmic SPOP rather than those
cell lines in which SPOP is absent from the cytoplasm.
Characterizations of the interactions between SPOP and inhibitors in vitro. The Tm
of SPOPMATH is shifted up to 3.5 C in the presence of the 20-fold compound 6lc
o
(Table 1), suggesting that 6lc binds to the substrate-binding MATH domain. We
further performed the nanoDSF assay under native conditions on the SPOP23-337
protein that contains both MATH and BTB domains. Compound 6lc thermally
stabilizes the SPOP23-337 protein by elevating the Tm up to 2.1 C even in an equal
molar ratio (Figure 5A). Although the negative control compound 6c exhibits little
effect on the thermal stability of SPOP in nanoDSF measurement (Figure 5B), it
appears that DMSO destabilizes SPOP and that the compound returns the Tm back
to its usual state in the absence of DMSO. A similar phenomenon is observed in the
conventional DSF screening, in which a fluorescent dye is required, although
compound 6lc significantly stabilizes SPOPMATH by shifting the Tm to 4 C (Figure 5C).
Again, the negative control 6c had minimal effect on the stability of SPOPMATH in the
conventional DSF assay (Figure 5D). Consistent with our previous conclusion that
o
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6
SPOP inhibitor 6b does not belong to the Pan Assay Interference Compound
1
4
(
PAINS), neither the conventional DSF performed with mixtures of just the dye and
compound 6lc yields any changes in fluorescence, nor does the nanoDSF experiment
conducted on compound 6lc and unrelated proteins yield any changes in
0
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fluorescence, for example, the BSA and ClpP caused no changes in thermal stability.
Instead of a fluorescence based DSF system, other biophysical methods, like Nuclear
Magnetic Resonance (NMR) titration and Surface Plasmon Resonance (SPR) should
also be better options in order to ensure that observed thermal effects are unlikely
due to the conjugated nature or the large concentration of compound 6lc.
Attenuation of signals in Carr-Purcell-Meiboom-Gill (CPMG) NMR titrations alongside
positive Saturation Transfer Difference (STD) signals are detected (Figures 5E and
S2A), which indicates that both SPOPMATH and SPOP in full-length interfere with the
state of compound 6lc. In addition, SPR measurement has established a moderate
affinity for inhibitor 6lc binding to the two SPOP proteins, which is around 30 μM
(Figure 5F and S2B). Lastly, a variety of SPOP inhibitors are observed to thermally
stabilize SPOP in the conventional DSF assay (Figure 5G). Collectively, we performed
different assays in order to confirm SPOP inhibitors binding to SPOP protein.
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Figure 5. Investigations on the binding of inhibitors to SPOP in vitro. (A) Effect of 6lc
on the thermal stability of SPOP23-337 in nanoDSF. (B) Effect of 6c on the thermal
stability of SPOPMATH in nanoDSF. (C) Effect of 6lc on the stability of SPOPMATH in the
conventional DSF assay. (D) Effect of 6c on the stability of SPOPMATH in the
conventional DSF. (E) NMR measurement of 6lc binding to SPOPMATH. The STD-NMR
spectrum for 200 μM 6lc is recorded in the presence of SPOPMATH. (F) Determination
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of the affinity of 6lc binding to SPOPMATH in SPR measurement. Graphs of equilibrium
RU responses versus compound concentrations are plotted. (G) Effect of SPOP
inhibitors on the stability of SPOPMATH in the conventional DSF assay. For nanoDSF
and conventional DSF assay, ΔTm for DMSO-treated SPOP protein were calculated
using a naive SPOP sample as the reference, while ΔTm data for the compound-
treated SPOP were reported using DMSO-treated SPOP as the reference.
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lc inhibits SPOP-PTEN interaction. In order to verify the cellular interaction
between 6lc and SPOP, we performed a Drug Affinity Responsive Target Stability
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(
DARTS) assay. As expected, the SPOP protein becomes protease-resistant in the
presence of 6lc (Figure 6A), indicating the direct interaction of 6lc with SPOP in cell
lysates of both A498 and OS-RC-2 cells. Next, we performed the Co-
immunoprecipitation (Co-IP) experiment in HEK293T cells in order to elucidate
whether 6lc could disrupt the SPOP-mediated protein interaction. As shown in Figure
6
B, the SPOP-binder 6lc is able to inhibit SPOP-binding with its substrate-protein
PTEN in a dose-dependent manner. Consequently, the abundance of ubiquitinated
PTEN protein is steadily reduced when HEK293T cells are exposed to the compound
6
lc in a concentration-dependent way (Figures 6C).
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Figure 6. Effect of 6lc on the interaction with SPOP and the disruption of the SPOP-
PTEN interaction in HEK293T cells. (A) Representative western blots for SPOP levels
in A498 and OS-RC-2 cells in DARTS. (B) Effect of 6lc on SPOP interaction with PTEN
in intact HEK293T cells in Co-IP assay. (C) Effect of 6lc on ubiquitination of PTEN in
HEK193T cells. The intensity in the western blot was read with ImageJ. All
experiments were performed in triplicate.
6
lc disrupts the oncogenic SPOP-signaling pathway in kidney cancer cells. Both
PTEN and DUSP7 are tumor suppressors that regulate cell proliferation and
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apoptosis through the cytoplasmic SPOP-signaling pathway in ccRCC. We have
previously demonstrated that SPOP inhibitors biochemically illuminate in cells the
therapeutic potential of blocking SPOP activity to stabilize PTEN and DUSP7, which
suppresses the AKT- and ERK-driven cancer-signaling pathways. Indeed, a steady
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accumulation of PTEN and DUSP7 proteins is observed in a dose-dependent manner
when A498 and OS-RC-2 cell lines are exposed to compound 6lc for 12 hr (Figure 7A).
Therefore, the accumulation of PTEN and DUSP7 in the cytoplasm subsequently
results in decreases of phosphorylated AKT and ERK in both ccRCC cell lines. The
total cellular abundance of AKT and ERK proteins in A498 and OS-RC-2 cells remains
constant in the presence of compound 6lc. Of note, Real-time quantitative PCR (RT-
qPCR) experiments showed that changes of PTEN and DUSP6 in A498 and OS-RC-2
ccRCC cell lines are not at the transcription level (Figure 7B). In addition, several
analogs of 6lc, namely compound 6lf, 6li, 6i, and 6l, also lead to the stabilization and
accumulation of PTEN protein in OS-RC-2 cells (Figure 7C).
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Figure 7. Effect of SPOP inhibitor on the oncogenic SPOP-signaling pathway in kidney
cancer cells. (A) Effect of 6lc on the protein levels in the SPOP-signaling pathway in
A498 and OS-RC-2 cell lines. (B) Effect of 6lc on the transcription of PTEN and DUSP7
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in A498 and OS-RC-2 cell lines. (C) Effect of SPOP inhibitors on the PTEN level in OS-
RC-2 cells. All experiments were performed in triplicate.
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CONCLUSION
We performed synthetic optimization based on the previously established chemical
scaffold to explore the SAR for potent SPOP inhibitors. We tested five sets of 6b
analogs to better understand which motifs in the molecule are important for SPOP
engagement. A nanoDSF-based high-throughput assay, that measures the thermal
stability of an interested protein, is applied as an initial screening of small-molecule
binders for SPOP protein. Then, the antiproliferative activities against A498 and OS-
RC-2 cell lines are evaluated, and the SAR is elucidated. Most amide analogs
thermally stabilize SPOP protein and exhibit antiproliferations in ccRCC cells, which
suggests that the nanoDSF is an option for the initial screening of SPOP inhibitors.
Compound 6lc exhibits strong inhibitory potency against both ccRCC cell lines with
IC of 2.1 μM and 3.5 μM, respectively. Additionally, compound 6lc significantly
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inhibits the colony formation for both A498 and OS-RC-2 cell lines, superior to our
previously reported 6b and other analogs tested. The performance of different
assays, including DSF, NMR titration, SPR analysis, and DARTS assay has confirmed
that inhibitor 6lc directly interacts with the SPOP protein in vitro and in cell lysates.
Lastly, an in-depth mechanistic study has revealed that compound 6lc disrupts SPOP-
substrate protein interaction in both ccRCC cell lines, and as a consequence leads to
the stabilization and accumulation of tumor suppressors PTEN and DUSP7 and
reduced abundances of phosphorylated AKT and ERK in the downstream. Taken
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together, we have demonstrated that the SPOP inhibitor 6lc represents a new lead
for further improvement to sufficiently target the cytoplasmic SPOP-signaling
pathway in ccRCC, which stands as a promising strategy to combat kidney cancer.
Although SPOP has also been shown to be a tumor suppressor in prostate cancer, it
is important to note that inhibition of SPOP may not be a strategy for all cancers.
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EXPERIMENTAL SECTION
Chemistry. Unless otherwise stated, all solvents and reagents were purchased from
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commercial sources and used as received. HNMR and CNMR spectra were
recorded with a Varian-MERCURY Plus-400, 500, and 600 NMR spectrometer at rt.
NMR spectra were calibrated to the solvent signals of DMSO-d , TFA-d, MeOD, or
6
CDCl . Chemical shifts are reported in ppm (δ scale) as referenced to TMS and
3
coupling constant (J) values are reported in hertz (Hz). Data are presented as follows:
chemical shift, multiplicity (s = singlet, d = doublet, dd = doublet of doublet, t =
triplet, q = quartet, m = multiplet, br = broad), J in Hz, and integration. Flash-column
chromatography was performed using silica gel (230–400 mesh). Analytical TLC was
performed on silica gel plates and visualized under ultraviolet light (254 nm). The
purity and LRMS of all the synthesized compounds were determined by LCMS
(Agilent 1200LC/G6110A, XBridge, 4.6 × 50 mm, 3.5 μm, 50 °C, flow rate = 1.8
mL/min) with aqueous CH CN and water (10 mM Ammonium hydrogen carbonate or
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.01% TFA). The purities of all target compounds are >95% in HPLC.
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Synthetic procedure for compound 2. A mixture of 3-methylpyridin-2-amine 1 and
o
diethyl malonate was stirred at 220 C for 2 hr. The generated ethanol was distilled
during reaction. The residue was recrystallized from ethanol to give product 2.
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Synthetic procedure for compound 3. To a cool DMF (144 mL) POCl (44 g, 288
3
o
mmol, 4.0 eq) was added dropwise. After the mixture was stirred for 30 min at 0 C,
compound 2 (11.8 g, 72 mmol) was added slowly. The reaction was continued at 0 oC
for 1 hr and at rt for 1 hr, and then was refluxed for another 1 hr. The mixture was
poured into ice water and stirred for 1 hr. The precipitate was filtered and washed
thoroughly with cold water. After a period of vacuum drying, compound 3 (10.2 g,
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3.8% yield) was collected. H NMR (400 MHz, DMSO-d ) δ 10.25 (s, 1H), 9.08 – 8.99
6
(m, 1H), 8.25 (d, J = 6.8 Hz, 1H), 7.58 (t, J = 7.0 Hz, 1H), 2.53 (s, 3H).
Synthetic procedure for compound 4. A mixture of intermediate 3 (446 mg, 2 mmol,
.0 eq) and corresponding amine (8 mmol, 4.0 eq) in EtOH (4 mL) was stirred at rt
overnight in the presence of anhydrous Na SO (100 mg). The precipitate was
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2
4
filtered and washed with EtOH. The solid was re-dissolved in CH Cl and filtered in
2
2
order to remove Na SO . Compound 4 was obtained after the evaporation of the
2
4
solvent under reduced pressure in good yields. Compounds 4k and 4p were directly
used in the next step without purification or NMR data collection.
(
E)-9-methyl-2-((4-methylphenethyl)amino)-3-(((4-
methylphenethyl)imino)methyl)-4H-pyrido[1,2-a]pyrimidin-4-one (4d). White solid
1
(400 mg, 46% yield). H NMR (400 MHz, CDCl ) δ 10.73 (s, 1H), 8.88 (s, 1H), 8.81 (d, J
3
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= 6.4 Hz, 1H), 7.49 (d, J = 6.7 Hz, 1H), 7.18 (dd, J = 8.2, 2.3 Hz, 4H), 7.14 (d, J = 6.0 Hz,
4
1
H), 6.79 (t, J = 7.0 Hz, 1H), 3.88 – 3.72 (m, 4H), 2.94 – 2.87 (m, 2H), 2.85 (dd, J =
5.5, 8.1 Hz, 2H), 2.49 (s, 3H), 2.35 (s, 3H), 2.34 (s, 3H).
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(
E)-2-((4-methoxyphenethyl)amino)-3-(((4-methoxyphenethyl)imino)methyl)-9-
methyl-4H-pyrido[1,2-a]pyrimidin-4-one (4e). White solid (452 mg, 48% yield). 1H
NMR (400 MHz, CDCl ) δ 10.72 (s, 0H), 8.87 (s, 0H), 8.80 (d, J = 6.9 Hz, 0H), 7.48 (d, J
3
=
6.7 Hz, 0H), 7.20 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 6.93 – 6.84 (m, 1H), 6.77
(t, J = 7.0 Hz, 0H), 3.91 – 3.73 (m, 10H), 2.88 (t, J = 7.4 Hz, 2H), 2.84 (dd, J = 14.7, 7.4
Hz, 2H), 2.48 (s, 3H).
(
E)-2-((4-fluorophenethyl)amino)-3-(((4-fluorophenethyl)imino)methyl)-9-methyl-
1
4
H-pyrido[1,2-a]pyrimidin-4-one (4f). White solid (473 mg, 53% yield). H NMR (500
MHz, CDCl ) δ 10.61 (s, 1H), 8.83 (s, 1H), 8.78 (d, J = 6.7 Hz, 1H), 7.47 (d, J = 6.1 Hz,
3
1
3
H), 7.24 – 7.11 (m, 4H), 6.98 (dt, J = 12.8, 8.5 Hz, 4H), 6.77 (t, J = 6.7 Hz, 1H), 3.86 –
.67 (m, 4H), 2.87 (t, J = 6.9 Hz, 2H), 2.82 (t, J = 6.7 Hz, 2H), 2.46 (s, 3H).
(
E)-2-((4-chlorophenethyl)amino)-3-(((4-chlorophenethyl)imino)methyl)-9-methyl-
1
4H-pyrido[1,2-a]pyrimidin-4-one (4g). White solid (556 mg, 58% yield). H NMR (400
MHz, CDCl ) δ 10.60 (s, 1H), 8.85 (s, 1H), 8.81 (d, J = 6.7 Hz, 1H), 7.50 (d, J = 6.7 Hz,
3
1
H), 7.28 (ddd, J = 10.8, 5.7, 2.1 Hz, 5H), 7.15 (dd, J = 15.3, 8.3 Hz, 4H), 6.80 (t, J = 7.0
Hz, 1H), 3.87 – 3.71 (m, 4H), 2.88 (t, J = 7.2 Hz, 2H), 2.83 (t, J = 7.1 Hz, 2H), 2.48 (s,
H).
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(E)-2-((4-bromophenethyl)amino)-3-(((4-bromophenethyl)imino)methyl)-9-methyl-
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4
H-pyrido[1,2-a]pyrimidin-4-one (4h). White solid (466 mg, 41% yield). H NMR (400
MHz, CDCl ) δ 10.58 (s, 1H), 8.85 (s, 1H), 8.80 (d, J = 7.0 Hz, 1H), 7.50 (d, J = 6.7 Hz,
3
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1H), 7.43 (dd, J = 12.1, 5.4 Hz, 4H), 7.10 (dd, J = 13.5, 8.3 Hz, 4H), 6.79 (t, J = 7.0 Hz,
1
H), 3.78 (dt, J = 14.4, 7.1 Hz, 4H), 2.86 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 7.0 Hz, 2H), 2.47
(s, 3H).
(
E)-2-((3-fluorophenethyl)amino)-3-(((3-fluorophenethyl)imino)methyl)-9-methyl-
1
4H-quinolizin-4-one (4i). White solid (339 mg, 38% yield). H NMR (600 MHz, CDCl3)
δ 10.62 (s, 1H), 8.84 (s, 1H), 8.79 (d, J = 7.0 Hz, 1H), 7.48 (d, J = 6.6 Hz, 1H), 7.28 –
7
.20 (m, 2H), 7.01 (d, J = 7.5 Hz, 1H), 6.99 – 6.94 (m, 2H), 6.94 – 6.87 (m, 3H), 6.78 (t,
J = 6.9 Hz, 1H), 3.80 (m, 4H), 2.90 (t, J = 7.3 Hz, 2H), 2.85 (t, J = 7.0 Hz, 2H), 2.47 (s,
H).
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(
E)-2-((2-fluorophenethyl)amino)-3-(((2-fluorophenethyl)imino)methyl)-9-methyl-
1
4
H-pyrido[1,2-a]pyrimidin-4-one (4j). White solid (552 mg, 62% yield). H NMR (400
MHz, CDCl ) δ 10.47 (s, 1H), 8.66 (d, J = 1.0 Hz, 1H), 8.58 (d, J = 7.0 Hz, 1H), 7.27 (d, J
3
=
6.7 Hz, 1H), 7.08 – 6.94 (m, 4H), 6.95 – 6.79 (m, 4H), 6.56 (t, J = 6.9 Hz, 1H), 3.73 –
3.52 (m, 4H), 2.76 (dt, J = 19.0, 7.1 Hz, 4H), 2.27 (s, 3H).
(
E)-9-methyl-2-((2-(thiophen-2-yl)ethyl)amino)-3-(((2-(thiophen-2-
yl)ethyl)imino)methyl)-4H-pyrido[1,2-a]pyrimidin-4-one (4l). Yellow solid (532 mg,
1
6
3% yield). H NMR (400 MHz, CDCl ) δ 10.61 (s, 1H), 8.70 (s, 1H), 8.61 (d, J = 6.9 Hz,
3
1H), 7.30 (d, J = 6.0 Hz, 1H), 6.98 (dd, J = 11.7, 5.1 Hz, 1H), 6.84 – 6.74 (m, 1H), 6.71
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