A covalent p97/VCP ATPase inhibitor can overcome resistance to CB-5083 and NMS-873 in colorectal cancer cells

Article abstract of DOI:10.1016/j.ejmech.2020.113148

Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multi

Full text of DOI:10.1016/j.ejmech.2020.113148

European Journal of Medicinal Chemistry 213 (2021) 113148
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
A covalent p97/VCP ATPase inhibitor can overcome resistance to CB-
5083 and NMS-873 in colorectal cancer cells
,
,
,
, 1, 2
Gang Zhang a 1, Shan Li a 1, Feng Wang a 1, Amanda C. Jones b
,
Alexander F.G. Goldberg b, Benjamin Lin b, Scott Virgil b, Brian M. Stoltz b
,
,
***
Raymond J. Deshaies a
**, Tsui-Fen Chou a
,
c
,
3
,
, d, *
a Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, United States
b Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, 91125, United States
c Howard Hughes Medical Institute, Chevy Chase, MD, 20815, United States
d Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA, 91125, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA
ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins
involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-
throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding
domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first
used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit
ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97
Received 22 November 2020
Received in revised form
16 December 2020
Accepted 28 December 2020
Available online 2 January 2021
Keywords:
P97
dependent and independent degradation. We selected a
b-nitrostyrene scaffold to further study the
structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of
pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-
tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18
(N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent
Covalent inhibitor
ATPase activity
Organic synthesis
Docking
Anti-proliferative activity
Proteomics
selectivity in an in vitro ATPase activity assay: IC50 of 0.6 mM, 300 mM, and 100 mM for wild type p97, yeast
Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of
Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified
and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with
Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and
hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA.
Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and
PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was
inhibited by PPA with IC50 of 2.7 mM, 6.1 mM, and 3.4 mM, respectively. In addition, PPA is able to inhibit
proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic
analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways
such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we
have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future
exploration to improve the potency of p97 inhibitors with different mechanisms of action.
© 2021 Elsevier Masson SAS. All rights reserved.
* Corresponding author. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, United States.
** Corresponding author. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, United States.
*** Corresponding author.
E-mail addresses: stoltz@caltech.edu (B.M. Stoltz), rdii2003@gmail.com (R.J. Deshaies), tfchou@caltech.edu (T.-F. Chou).
These authors made equal contributions to this work.
Current address:Department of Chemistry, Box 7486, Wake Forest University.
Current address:Amgen Research, Thousand Oaks, CA, USA.
1
2
3
https://doi.org/10.1016/j.ejmech.2020.113148
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
1. Introduction
grade inhibitor of p97, is in phase I clinical trials for Acute Myeloid
Leukemia or Myelodysplastic Syndrome [25]. One of the major
challenges of targeted cancer therapy is the emergence of drug-
resistant mutations in tumor cells leading to loss of treatment
effectiveness. Mutations in p97 that cause resistance to CB-5083
have been identified [23]. Availability of different p97 inhibitors
with various binding modes may help overcome this issue.
Recently, LC-1028 was reported as a covalent p97 inhibitor with a
Michael acceptor consisting of a carbonyl group conjugated with a
methyl-capped acetylene group [26]. LC-1028 showed anti-
proliferative activity in MIA PaCa-2 cells with an EC50 value of
ATPases
associated
with
diverse
cellular
activities
(AAA þ proteins) are enzymes sharing a common conserved
module of approximately 230 amino acid residues [1] and are
functionally diverse protein families belonging to the
AAA
þ
protein superfamily of ring-shaped P-loop NTPases.
AAA þ proteins catalyze the hydrolysis of a phosphate bond in
adenosine triphosphate (ATP), releasing adenosine diphosphate
(ADP), inorganic phosphate, and energy to facilitate a variety of
cellular processes that are essential for life, including protein
folding [2], intracellular transport [3], protein degradation [4],
initiation of DNA replication [5], DNA repair [6], DNA remodeling
[7], and ion transport [8] .
0.436 mM. LC-MS/MS suggested LC-1028 conjugates to C522, indi-
cating that LC-1028 binds to the D2 domain by a covalent bond; the
docking model was consistent with this result. However, the
selectivity of LC-1028 against WT-97 over other AAA þ ATPases
remains unclear. Although there is a much research on the inhibi-
tion of p97 and clinical trials are ongoing to evaluate CB-5339, no
drug has been approved by the FDA to target p97.
Nerviano Medical Sciences and Genentech discovered the allo-
steric inhibitor NMS-873, an alkylsulfanyl-1,2,4-triazole analog that
binds in a tunnel between the D1 and D2 domains [27,28]. NMS-
873 inhibited the ERAD pathway and led to the accumulation of
misfolded proteins in the ER [29]. UPCDC30245 is another allosteric
p97 inhibitor with an IC50 of 27 nM, which reversibly binds at the
interface of the D1 and D2 domains as determined by cryo-electron
microscopy [30]. Eeyarestatin I (EerI) serves as a prodrug to convert
the 5-nitrofuran-2-yl (NFC) group into a reactive metabolite
involved in inhibition of p97-dependent protein degradation in
cells even though the exact mechanism of the active metabolite still
remains unknown [31,32]. NMS-859 is a covalent p97 inhibitor,
p97, also known as valosin-containing protein (VCP) and Cdc48
(cell division cycle protein 48) in Saccharomyces cerevisiae, Ter94
(transitional endoplasmic reticulum ATPase) in Drosophila mela-
nogaster and VAT (VCP-like ATPase) in Thermoplasma acidophilum,
is a hexameric type II AAA þ ATPase. p97 is involved in a wide range
of cellular functions, including endoplasmic reticulum-associated
degradation (ERAD), membrane fusion, nuclear factor kappa-
light-chain-enhancer of activated B cells (NF-kB) activation, and
chromatin-associated processes, which are regulated by ubiquiti-
nation [9]. p97 consists of two AAA ATPase domains in tandem, D1,
and D2, respectively. A short polypeptide linker (D1-D2 linker)
connects the two ATPase domains while the N-D1 linker joins the
D1 domain to a large amino-terminal domain (N-domain). The
carboxyl-terminus of the D2 domain features a short tail containing
~40 residues. Interaction of p97/Cdc48 with its partners is mostly
mediated by the N-domain, although a few proteins bind p97/
Cdc48p via the C terminus [10,11]. Both D1 and D2 domains are
homologous in sequence and in structure [12]. The D1 domain has
lower intrinsic ATPase activity than does the D2 domain [13], while
the D2 domain was thought to underlie p97 function as a mecha-
nochemical transducer, which contributed most of the ATPase ac-
tivity [14]. Protein substrates released by p97 can be degraded by
the 26S proteasome [15,16]. In addition, p97 is involved in another
form of protein degradation and recycling, namely autophagy [17].
The expression levels of p97 are upregulated in many cancers, such
as colorectal cancer, pancreatic cancer, thyroid cancer, squamous
cell carcinoma, breast cancer, osteosarcoma, gastric carcinoma, and
lung cancer [18]. Therefore, p97 is considered a potential anticancer
target.
Currently, there are several p97 inhibitors reported (Fig. 1),
classified as either ATP-competitive or allosteric inhibitors. The
ATP-competitive inhibitor DBeQ (N2, N4-Dibenzylquinazoline-2,4-
diamine) mainly targets the D2 domain. DBeQ blocks many bio-
logical processes, including degradation of ubiquitin-fusion
degradation and ERAD pathway substrates, as well as autophago-
some maturation [19e21]. Two DBeQ derivatives with IC50 values of
~100 nM, ML240 and ML241, have been developed. ML240 stimu-
lates the accumulation of LC3-II and rapidly mobilizes the execu-
tioner caspases 3 and 7. ML240 also has broad antiproliferative
activity toward the NCI-60 panel of cancer cell lines and synergizes
with the proteasome inhibitor MG132 to kill multiple colon cancer
cell lines. Both ML240 and ML241 have low off-target activity to-
ward a panel of protein kinases and central nervous system targets
[22]. CB-5083, derived by structure-activity relationship of the
featuring an electrophilic
modifies Cys522 in the D2 active site with an IC50 of 0.37
MSC1094308 [33] and 2-aminopyridine indole amide [34] are
reported as two allosteric p97 inhibitors with IC50 values of 7.2
and 0.5 M, respectively. MSC10944308, with low biochemical
potency, showed cellular efficacy at 10 M. The 2-aminopyridine
indole amide exhibited excellent physicochemical properties,
including solubility (330 M in PBS), bidirectional permeability
(710 and 460 nm/s), and microsomal stability (human T1/
2 > 60 min). (10,11)-Dehydrocurvularin [35] and Withaferin A 27-
Acetate [36] covalently modify p97. (10,11)-Dehydrocurvularin
inhibited p97 with an IC50 value of 15.3 9.9 M by covalent bond
with Cys522 of p97. Withaferin A 27-Acetate exhibited inhibitory
activity against wild-type p97 with an IC50 value of 21 M, but it
a
-chloroacetamide, which selectively
mM [28].
mM
m
m
m
D
D
m
m
didn’t show inhibition against the C522A mutant. However, With-
aferin A 27-Acetate exhibited anti-proliferative effects in cancer cell
lines at 10-fold lower concentration, suggesting additional modes
of action. Clotrimazole [37], Oxaspirol B [38], and 50-I-Fuligocandin
B [39] exhibited p97 inhibition activity with IC50 values of 12
mM,
31.2 3.0 M and 7.0 M, respectively. However, their mechanisms
m
m
of action are unclear. Herein, we report the design, optimization,
and biological evaluation of a covalent p97 inhibitor with the po-
tential to be a specific and useful tool to uncover p97’s function and
to be further developed to treat cancers.
2. Results and discussion
2.1. Search for potential irreversible kinase inhibitors to inhibit p97
in vitro and in cells
ML240 scaffold, is
a selective, ATP-competitive, and orally
bioavailable p97 inhibitor, which activates the apoptotic arm of the
unfolded protein response and exhibits antitumor activity in
several hematological and solid tumor models. CB-5083 entered
phase I clinical trials for multiple myeloma in 2015 [23,24]. How-
ever, the trial was terminated due to toxicity caused by an off-target
effect. Its successor CB-5339, as the second-generation clinical-
High throughput screens (HTS) identified multiple hits con-
taining electrophilic groups with sub-micromolar IC50 values for
inhibiting ATPase activity of purified p97 in vitro and turnover of a
p97-dependent cellular reporter [21]. C522A p97 is less sensitive to
these electrophilic compounds [21,40]. One of the hits, an aryl
2

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
Fig. 1. Chemical Structure of Known p97 Inhibitors.
alkynyl ketone (Compound 1, JFD02342), inhibited ATPase activity
with IC50 of 0.2 M. However, it also inhibited the turnover of both
to the nitro styrene moiety (compound 9). The requirement for the
nitro group and the relative insensitivity of the remainder of the
scaffold to modification suggested that the primary mechanism of
action was a covalent reaction with Cys522 in the D2 domain active
site. The poor activity of Compound 2 towards the C522A mutant
and its sensitivity to DTT indicated that the electrophilic attack of
Cys522 was indeed critical for the potency of Compound 2 [41].
Compound 2 was first reported to be a selective Syk inhibitor [43]
with submicromolar IC50; however, our results indicated that it is a
potent p97 inhibitor in vitro and in cells, but we were unable to
obtain a selective analogue for inhibiting p97 in cells, which is likely
due to the high reactivity of the electrophilic moiety. We then
sought to design an irreversible scaffold by using x-ray crystal
structures of p97 (PDB code1e32).
m
p97-dependent (UbG76V-GFP) and independent reporters (ODD-
Luc) (Table 1). To search for irreversible p97 inhibitors, we evalu-
ated several characterized kinase inhibitors with our in vitro and
cell-based p97 HTS assays [41]. Compound 2 is a spleen tyrosine
kinase (Syk) inhibitor [42,43], compounds 4 (cmk), and 5 (fmk) are
halomethylketone pyrrolopyrimidine-based p90 ribosomal protein
S6 kinase (RSK) inhibitors [44], and compound 6 is a 6-acrylamido-
4-anilinoquinazoline-based inhibitor against epidermal growth
factor receptor (EGFR) [45]. Compounds 1e3 demonstrated sub-
micromolar IC50, compound 5 was 10-fold less active than the
corresponding chloro-substituted compound 4, and compound 6
exhibited IC50 values about 10e18
mM (Table 1). Compound 2 (3,4-
methylenedioxy- -nitrostyrene) [41] was chosen for more detail
b
analysis due to its lower IC50 values in both in vitro ATPase and in
2.2. Structure-based drug design to develop a covalent inhibitor of
p97
cell UbG76V-GFP degradation assays.
To examine structure-activity relationships of the
b-nitro-
styrene scaffold, we obtained five and prepared five analogues
(compounds 7e16, Table 2). Replacing the nitro group with car-
boxylic acid (compound 7) led to complete loss of activity, therefore
suggesting the nitro group is critical for the observed inhibition.
The only other modification that affected both assays by more than
3-fold was the placement of a hydroxyl group in para-substitution
To develop covalent inhibitors of p97, we focused our attention
on the D2 ATPase domain. This domain has a cysteine residue at
position 522, the side chain of which projects into the active site
nearby the amino group (NH2) attached at the C6 position of the
purine of a bound ADP-aluminum fluoride complex (Fig. S1a) [46].
We reasoned that an ATP analog carrying an electrophilic
3

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
Table 1
Apparent IC50 values of known irreversible inhibitors against p97 and its reporters.
Compounds
Structures
Apparent IC50
UbG76V-GFP
3.4 1.0
(m
M)a
ODD-Luc
5.2 1.8
p97
1b (JFD02342)
0.2 0.02
2b
(
b
-nitrostyrene)
1.6 0.4
3.7 0.4
3.9 1.0
NDc
1.7 0.5
3 (EerI)
>30
4 (cmk)
5.8 1.1
4
2
3.9 0.9
5 (fmk)
47 10
39
11
3
5
>30
6
18
4
10
5
a
Measurements were carried out in triplicate, and variance is expressed as the standard deviation.
Data for compounds 1 and 2 were adapted from Refs. [21,41], respectively.
ND: Not Determined.
b
c
substitution at C6 might react with this cysteine residue and
2.3. Biochemical characterization of PPA (18)
inactivate p97. It is noteworthy that a cysteine at position 522 is not
essential for p97 ATPase activity, and in fact the budding yeast
ortholog of p97, Cdc48, does not have cysteine in this position.
Indeed, the presence of cysteine at this position in members of the
AAA ATPase family is variable: the D1 domain of NSF has a cysteine
in the equivalent position, whereas five of the six AAA ATPase
subunits of the 26S proteasome do not (Fig. S1b). To develop an
inhibitor capable of reacting with this cysteine, we selected PP
(compound 17) as the starting point and optimized this scaffold in a
variety of ways in terms of the binding mode of the Src inhibitor
pyrazolopyrimidines (PPs) (Fig. 2) [47,48]. Specifically, we attached
various electrophilic groups to the C4 position, including acryl-
amides (compounds 18, 19, 22, 23, 24, 27, and 28), and a chlor-
oacetamide (compound 20). As negative controls, we prepared
compounds that retained an amino group attached at C4 (com-
pounds 17, 21, 25, and 26). All compounds demonstrated inhibitory
activity in both ATPase and UbG76V-GFP turnover assays except for
the control compounds (Table 3). The most potent inhibitor was
compound 18 (PPA), which contains benzene at C3 and a single
acrylamide at C4. PPA was 20-fold less potent at inhibiting the p97-
independent reporter. Therefore, we selected PPA for a more
detailed study.
To determine if inhibition by PPA requires a reactive thiol, we
determined inhibition of ATPase activity in the presence of
dithiothreitol (DTT) and cysteine during the preincubation. The
data in Fig. S1c demonstrated that the inhibitory effect of PPA could
be counteracted by the inclusion of either cysteine or DTT. To
evaluate whether PPA modifies cysteine 522, inhibited p97 was
digested with trypsin, and peptides were fractionated by HPLC and
analyzed by tandem mass spectrometry. The PPA-modified peptide
(N14) from the digested p97 sample was eluted using PPA-modified
N15-labeled peptide (H2N-GVLFYGP*P*GCGK-OH, where * in-
dicates N15 labeled residues) as an internal control (Fig. S2). Tan-
dem mass spectrometry identified Cys522 as the site of
modification, thus confirming the formation of a covalent bond
between PPA and p97. To further address whether Cys522 is
necessary for inhibition of p97 by PPA, we constructed two mutants
in which Cys522 was converted to either alanine or threonine.
Furthermore, we constructed a mutant of yeast Cdc48 in which the
residue analogous to Cys522, Thr532, was converted to cysteine.
Mutation of Cys522 in p97 to either alanine or threonine decreased
sensitivity to PPA by more than 100-fold (Table 4). On the other
hand, yeast Cdc48, which normally is quite resistant to PPA, became
4

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
Table 2
Apparent IC50 values of compound 2 analogues against p97 and its reporters.
Compounds
Structures
Apparent IC50
UbG76V-GFP
>30
(mM)
ODD-Luc
p97
7
>30
>30
8
1.9 0.9
1.8 0.9
3.1 1.8
9
6.8 2.5
5.7 1.5
5
0.8
13
5
10
2.3 0.9
1.6 0.8
3.6 2.8
11
12
4.3 1.6
5.7 2.8
3.4 1.8
2.0 0.4
10
3
13
14
15
2.9 0.7
1.7 0.7
2.7 1.0
3.0 1.0
4.0 1.4
2.1 0.3
0.7 0.3
1.2 0.3
2.4 0.6
16
1.8 0.8
2.0 0.9
1.7 0.5
Fig. 2. Structures of the pyrazolo[3,4-d]pyrimidine analogues designed for p97 inhibition.
Table 3
nearly 30-fold more sensitive to the compound upon the intro-
duction of cysteine in place of Thr532. Together, these data indicate
that Cys522 is both necessary and sufficient to confer sensitivity to
PPA.
Apparent IC50 values of the pyrazolo[3,4-d]pyrimidine analogues against p97,
T532C-Cdc48, and the cellular reporters.
Compounds
Apparent IC50
UbG76V-GFP
(
m
M)a
ODD-Luc
p97
T532C-Cdc48
2.4. Docking study of PPA (18)
17
18 (PPA)
19
20
21
22
23
24
25
>30
1.6 0.5
5.4 1.6
31
>30
2.0 0.9
2.5 0.3
3.6 0.5
>30
>30
2.1 0.6
6.1 0.7
>30
35 10
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>100
>100
14
26
35
>100
24
22 11
ND
ND
ND
0.6 0.2
1.1 0.7
4.5 3.5
>100
5
6
8
Although we have confirmed the covalent binding of PPA to
Cys522 in Domain 2, it is still unclear whether other interactions
between PPA and WT-p97 occur. In order to understand the binding
mode of PPA in the active site, we performed the covalent docking
study by AutoDock 4.2.6 [49]. In addition to the covalent bonding to
Cys522 between the terminal carbon atom from the acryl amido
group and the thiol group, there were two hydrogen bond in-
teractions between Gly480 and 7-NH, and Gly521 and the amido
NH, respectively (Fig. 3A and B). In addition, some residues in the
active site, such as Ile479, Leu526, Gly684, Ala685, and Thr688
showed hydrophobic interactions with PPA (Fig. 3A).
3
3
1
7
1.3 0.7
10
>50
>50
2.8 1.2
0.9 0.2
6
26
27
28
ND
ND
ND: not determined.
a
Measurements were carried out in triplicate, and variance is expressed as the
standard deviation.
5

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
Table 4
known p97 inhibitors, CB-5083, NMS-873, and DBeQ. As shown in
Fig. 5A, all compounds inhibited proliferation of all three cancer
lines. CB-5083 showed the most potent anti-proliferative activity.
Cys522-mediated p97 inhibition by PPA (18) in vitro.
Enzyme
Apparent IC50 (mM)
The IC50 of PPA to HCT116, HeLa, and RPMI8226 were 2.7
mM,
WT-p97
C522A-p97
C522T-p97
Yeast Cdc48
T532C-yeast Cdc48
Hamster NSF
Human 19S ATPasea
0.62 0.25
110 33
82 45
6.1 M, and 3.4 M respectively. PPA has comparable potency as
m
m
DBeQ and less potent compared to CB-5083 and NMS-873. PPA’s
cellular activity was consistent with its enzymatic activity against
p97. Furthermore, we evaluated its potency toward CB-5083
resistant (CB-R) and NMS-873 resistant (NMS-R) HCT116 cell lines
[24], as shown in Fig. 5B, PPA and DBeQ have similar anti-
proliferative activities for all three HCT116 lines. CB-5083 is 72-
fold less effective in CB-R, whereas NMS-873 is 4.5-fold less effec-
tive in NMS-R. The two NMS-873 resistant cell lines we isolated are
A530T heterozygous, so the fold resistance is not as high as the
homozygous A530T made using CRISPR as described previously
[54].
376 95
14
5
105 31
75 19
a
Human Rpt3 contains a cysteine in Walker A motif.
2.5. PPA does not inhibit NSF and 26S proteasome and blocks
degradation of p97 substrate irreversibly
To further address the specificity of PPA, we tested its activity
against two other AAA ATPases, NSF, and the 19S regulatory par-
ticles. Although NSF is known to be sensitive to thiol-reactive
agents, it was more than 100-fold less sensitive to PPA than p97
(Fig. 4A, Table 4). Likewise, inhibition of ATP hydrolysis by the 19S
regulatory particles was observed only at high concentrations of
2.7. Quantitative proteomic analysis of PPA treated HCT116 human
colon cancer cells
Label-free quantification using the Orbitrap Eclipse LC-MS/MS
PPA (IC50 ¼ 75
mM). The results with NSF and the 19S regulatory
was employed to evaluate the effect of PPA (15 mM or 30 mM for
particles are noteworthy because both the active D1 domain of NSF
and the Rpt3 subunit of the 19S regulatory particles have cysteine
in the position analogous to cysteine 522 of p97 (Fig. S1b). More-
over, PPA did not inhibit the hydrolysis of a fluorogenic proteasome
substrate, LLVY-AMC, with the affinity-purified 26S proteasome
from Hek293 cells [50] (Fig. 4B). We used previously established
washout assay using UbG76V-GFP reporter to demonstrate PPA
6 h) on the protein level changes in HCT116 cells. Two independent
biological replicates were performed for each group. The proteome
analysis using PD 2.4 software identified a total of 5924 proteins
from all control and PPA-treated groups. The normalized abun-
dance was used for the following analysis (Fig. S3). We identified
291 proteins change in 15
553 proteins change in 30
m
m
M PPA-treated samples (Fig. 6A), and
M PPA-treated samples (Figs. 6B and
irreversibly block degradation of UbG76V-GFP in cells [41]. UbG76V
GFP was degraded after washing out MG132 but it remains stable
with PPA treated cells (Fig. 4C). Similarly, we showed that PPA block
-
7A) by limma analysis (p < 0.05). The Venn diagram shows that
the level of 58 proteins significantly increased (Fig. 6C, Table S3A)
and 46 significantly decreased (Fig. 6D, Table S3B) in both treat-
ment groups. By overlapping with known p97 interaction data from
BioGRID database, we found 33 of them significantly increased
(Fig. 6C) and 42 of them significantly decreased (Fig. 6D) in either
degradation of an ERAD substrate, TCRa-GFP (a-chain of the T-cell
receptor fused to GFP) [51]. We included MG132 (a reversible
proteasome inhibitor), YU101 (a covalent proteasome inhibitor)
[52], PYR41(a ubiquitin E1 inhibitor) [53], and DBeQ (a reversible
p97 inhibitor) [21] as positive controls.
15 or 30 mM treatment groups. Notably, there are 15 known p97
interacting proteins also showed differentially change in both
treatment groups and the log2 fold change values are shown in
Table 5. For example, SQSTM1 (also known as p62) [55], a ubiquitin
binding protein involved in autophagy, antioxidant response,
apoptosis, and regulation of endosomal trafficking, is increased in a
2.6. Anti-proliferative effect of PPA in cancer cells and in CB-5083,
NMS-873 resistant colorectal cancer
To explore anti-proliferative effects of these compounds, we
used three cancer cell lines and evaluated PPA together with three
concentration-dependent manner (logFC 0.42 vs 0.97, 15
mM vs
30 M). Another protein GOLGA3 [56], participating in transport of
m
Fig. 3. The docking study of PPA into WT-p97. (A) Two-dimensional presentation of the potential interactions between PPA and WT-p97. (B) Three-dimensional presentation of PPA
in the active site.
6

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
Fig. 4. PPA inhibits p97 ATPase activity selectively and irreversibly. (A) Titration curves of the in vitro ATPase assays for PPA inhibition of p97, NSF, and Cdc48. (B) PPA did not inhibit
human 26S proteasome activity. (C) Reversibility of PPA inhibition was determined using UbG76V-GFP by washing out test compound and monitoring decay of GFP signal in the
presence of cycloheximide for additional 17 h. (D) PPA block degradation of an ERAD substrate, TCR
washed and treated with cycloheximide plus compound for 60 min or 120 min. Samples were immunoblotted with anti-p97 as loading control and anti-GFP antibody to detect
TCR -GFP.
a-GFP stably expressed in HEK293 cells. Cells were incubated with MG132,
a
Fig. 5. Anti-proliferation activity of p97 inhibitors against (A) HCT116, HeLa, and RPMI8226 cell lines. (B) HCT116, CB-5083 resistant HCT116 cell line harboring D649A, T688A p97
mutant (CB-R) and NMS-873 resistant HCT116 cell line harboring A530T p97 mutant (NMS-R). Data are shown as Mean SEM taken from four replicate experiments.
protein, apoptosis, and positioning of the Golgi, is dose-
M vs 30 M).
These results provide valuable insights for further investigation
into the mechanisms by which PPA affects p97 function.
annotation enrichment analysis was carried out with the complete
human gene set as the background. We investigated the biological
process, molecular function, cellular component, and functional
pathways in the significantly enriched GO terms. The top fourteen
relevant changed biological processes are presented in Fig. 7B. The
analysis showed these DEPs are primarily related to protein bind-
ing, DNA binding and damage response, and mitochondrion. Some
p97-related processes, such as protein ubiquitination and ER to
Golgi transport vesicle membrane, were also identified, with other
processes linked to PPA’s anti-proliferation activity, such as p53
regulation, cell proliferation, and negative regulation of apoptosis.
Furthermore, protein-protein interaction (PPI) analysis was per-
formed using the STRING database to understand the interactions
between these DEPs in response to treatment with PPA in
dependently decreased (logFC ꢀ0.36 vs ꢀ0.70, 15
m
m
The 30
mM PPA-treated group had more significantly changed
proteins (~2 fold more than 15
mM PPA-treated group), which
suggests that this group might be more informative as to PPA
induced proteomic changes. Therefore, subsequent analyses
compared DMSO and 30
teins identified in this analysis was classified as the final differen-
tially expressed proteins (DEPs) between the control and 30
mM PPA-treated groups. A set of 110 pro-
mM
PPA-treated groups (listed in Table S2), with p-value<0.05 and fold
change >2 (up-regulated) or <0.5 (down-regulated). Subsequently,
the 110 DEPs were uploaded to the DAVID Database, and the
7

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
Fig. 6. Proteomic analysis of PPA treated HCT116 cell line. A-B) Volcano plots illustrate significantly differentially expressed protein in 15
HCT116 cells (fold change: PPA/DMSO). C-D) Venn diagrams of up-regulated protein (C) and down-regulated (D) proteins (p < 0.05) between 15
p97 interactors. The log2FC range of 15 M are ꢀ5.03 to ꢀ0.26 and 0.27 to 8.85.
M is ꢀ5.56 to ꢀ0.26 and 0.26 to 3.24 and the log2FC range of 30
m
m
M (A) and 30
mM (B) PPA treated
M, 30 M PPA-treated groups and
m
m
m
Fig. 7. A) Heatmap presentation of differentially regulated proteins (p < 0.05) of DMSO vs. 30
differentially expressed proteins [p < 0.05, and log2 (FC) > 1 or < ꢀ1].
mM PPA-treated groups. B) The biological process analysis of 30 mM PPA-treated group
HCT116 cell lines (Fig. S4). For all PPI networks, the minimum
required interaction score was set at medium confidence (0.400).
The PPI networks showed that the DEPs have several interactions
among themselves, suggesting these proteins are biologically
connected. For example, the protein HMOX significantly up-
regulated in both 15 and 30 M PPA-treated groups, was shown
to interact with many proteins, such as KEAP1, MSRA, MKLN1,
CDKN1A, and FTH1.
m
8

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
Table 5
Overlapped protein list of differentially expressed proteins by 15 mM and 30 mM PPA-treatment known to be regulated by p97. (p < 0.05).
Accession
Gene Symbol
Description
log2 (Fold Change)
15
m
M
30 mM
Q15149
P58107
Q13501
Q8TCF1
Q8TDN6
O43422
P46736
Q13586
P11279
O14519
Q14145
P50748
Q9Y679
Q08378
A0A087WY71
PLEC
Isoform 3 of Plectin
Epiplakin
Sequestosome-1
0.33
0.38
0.42
0.49
0.52
0.69
0.94
1.49
ꢀ0.59
ꢀ0.48
ꢀ0.46
ꢀ0.43
ꢀ0.39
ꢀ0.36
ꢀ0.33
0.58
0.64
0.97
0.42
0.70
0.68
0.83
1.65
ꢀ0.66
ꢀ0.47
ꢀ1.03
ꢀ0.87
ꢀ0.43
ꢀ0.70
ꢀ0.38
EPPK1
SQSTM1
ZFAND1
BRIX1
PRKRIR
BRCC3
STIM1
LAMP1
CDK2AP1
KEAP1
KNTC1
AUP1
AN1-type zinc finger protein 1
Ribosome biogenesis protein BRX1 homolog
52 kDa repressor of the inhibitor of the protein kinase
Lys-63-specific deubiquitinase BRCC36
Stromal interaction molecule 1
Lysosome-associated membrane glycoprotein 1
Cyclin-dependent kinase 2-associated protein 1
Kelch-like ECH-associated protein 1
Kinetochore-associated protein 1
Ancient ubiquitous protein 1
GOLGA3
AP2M1
Golgin subfamily A member 3
AP-2 complex subunit mu
3. Conclusion
0.040e0.063 mm) was used for flash chromatography. 1H and 13C
NMR spectra were recorded on either a Varian Mercury 500 spec-
trometer at 500 MHz and 125 MHz, respectively, or a Varian Mer-
cury 300 spectrometer at 300 or 75 MHz, respectively. 13C spectra
are referenced to residual CDCl3 (77.2). 19F NMR spectra were
recorded on a Varian 300 (at 282 MHz) and are reported relative to
To develop irreversible p97 inhibitors, we screened six well-
characterized kinase inhibitors by our in vitro and cell-based p97
HTS assays. Compounds 1 and 2 were chosen to further optimize
their structures to provide 10 compounds (compounds 7e16).
Intrigued by the p97 inhibition result of these compounds, we
designed and synthesized a series of irreversible p97 inhibitors by
introducing a Michael Acceptor on the N-6 position of ADP after
analysis of the crystal structure of the p97 complex with ADP-AlF3
(PDB code 1e32). PPA showed potent inhibition against p97 with
CFCl3
(d
0.0). Data for 1H and 19F NMR spectra are reported as fol-
ppm) (multiplicity, coupling constant (Hz),
lows: chemical shift (
d
integration). MS were acquired using an Agilent 6200 Series TOF
with an Agilent G1978A Multimode source in electrospray ioniza-
tion (ESI), atmospheric pressure chemical ionization (APCI) or
mixed (MM) ionization mode. Some compounds were synthesized
in this work and others were obtained from the in-house com-
pound library.
IC50 of 0.6 0.2 mM. Its irreversible mode of action was confirmed
by tandem mass spectrometry and a washout experiment in a p97
dependent degradation assay. An in-silico docking study was per-
formed to validate the feasibility of covalent binding to Cys522. In a
panel of Cys522-mediated p97 inhibition assays in vitro, PPA
showed up to 100-fold selectivity on wild-type vs. Cys522-mutated
p97 with IC50 of 0.62
inhibition against NSF and 26S proteasome. In addition, PPA
showed potent anti-proliferative effects on HCT116, HeLa, and
4.1.2. Synthesis of
b-nitro styrenes
b
-Nitro styrenes were prepared according to a literature pro-
0.25 mM. In addition, PPA did not show
cedure [57] with minor modifications See below for a representa-
tive procedure and Scheme 1 for syntesis of compound 8.
E-1,2-dimethoxy-4-(2-nitrovinyl)benzene (8). A round-bottom
flask fitted with a magnetic stir bar and a reflux condenser was
flame-dried under vacuum. After cooling to ambient temperature
under dry nitrogen, 3,4-dimethoxybenzaldehyde (0.419 g,
2.5 mmol, 1.0 equiv), ammonium acetate (0.170 g, 2.2 mmol, 0.9
equiv), and nitromethane (12.5 mL) were added, and the resulting
mixture was heated to reflux and maintained with stirring for 14 h.
The reaction mixture was concentrated under reduced pressure,
and the residue was recrystallized from ethanol to yield (8)
(0.362 g, 69% yield) as yellow-green flakes: 1H NMR (300 MHz,
RPMI8226 cells with IC50 values of 2.7 mM, 6.1 mM, and 3.4 mM,
respectively. Moreover, PPA can inhibit growth of CB-5083 and
NMS-873 resistant HCT116 cells. Furthermore, proteomic analysis
revealed PPA regulated bona fide p97 interacting proteins involved
in known p97 functional pathways such as protein ubiquitination
and ER to Golgi transport vesicle membranes. Taken together, these
observations suggest PPA could serve as a promising starting point
to develop an irreversible p97 inhibitor to treat cancer and can be a
valuable tool to overcome resistance to other p97 inhibitors with
different mode of actions such as ATP competitive and allosteric
mechanisms.
CDCl3)
d
7.97 (d, J ¼ 13.5 Hz, 1 H), 7.53 (d, J ¼ 13.5 Hz, 1 H), 7.18 (dd,
J ¼ 8.7, 2.1 Hz, 1 H), 7.01 (d, J ¼ 2.1 Hz, 1 H), 6.92 (d, J ¼ 8.7 Hz, 1 H),
3.94 (s, 3 H) 3.93 (s, 3 H); MS (EIþ) m/z 210.0 (M þ H)þ.
E-3-(2-nitrovinyl)pyridine (13). Prepared by the above proced-
ure (3-pyridylcarboxaldehyde, 0.25 mL, 2.7 mmol). Purified by
filtration through a short plug (SiO2, 50% EtOAc in hexanes) fol-
lowed by recrystallization from ethanol to yield (13) (0.112 g, 28%
4. Materials and methods
4.1. Chemical synthesis
yield) as orange needles: 1H NMR (300 MHz, CDCl3)
d 8.80 (d,
4.1.1. Material
J ¼ 2.1 Hz, 1 H), 8.72 (d, J ¼ 4.8, 1.5 Hz, 1 H), 8.01 (d, J ¼ 13.8 Hz, 1 H),
Reactions were performed in flame-dried glassware under an
argon or nitrogen atmosphere. Solvents were dried by passage
through an activated alumina column under argon. Triethylamine
(NEt3) was distilled from sodium hydride immediately prior to use.
Other commercial reagents were used as received. Thin-layer
chromatography (TLC) was performed using E. Merck silica gel 60
F254 precoated plates (0.25 mm) and visualized by UV fluorescence
quenching. Silica Flash P60 Academic Silica gel (particle size
7.87 (ddd, J ¼ 7.8, 2.1, 1.5 Hz, 1 H), 7.62 (d, J ¼ 13.5 Hz, 1 H), 7.41 (dd,
Scheme 1. Synthesis of compound 8.
9

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
J ¼ 7.8, 4.8 Hz,1 H); HRMS (MM: ESI-APCI) calc’d for C7H7N2O2 [M þ
H]þ: 151.0502, found 151.0503.
NMR (300 MHz, DMSO‑d6): d 11.04 (s, 1 H), 8.81 (s, 1 H), 7.61-7.56
(m, 2 H), 7.41-7.29 (m, 3 H), 6.37 (dd, J ¼ 17.2, 10.2 Hz, 1 H), 5.86 (dd,
J ¼ 17.2,1.6 Hz,1 H), 5.65 (dd, J ¼ 10.2,1.6 Hz,1 H),1.82 (s,9 H). HRMS
(EIþ) m/z calc’d for C18H19N5O [M]þ: 321.1590, found 321.1598. The
13C NMR data with good quality could not be obtained due to poor
signal-to-noise as a result of a peak broadening presumed to be a
result of the dynamic process that was not investigated further due
to the high reactivity of 18.
E-1-nitro-3-(2-nitrovinyl)benzene (14). Prepared by the above
procedure (3-nitrobenzaldehyde, 0.389 g). Purified by flash chro-
matography (SiO2, 17% EtOAc in hexane), followed by recrystalli-
zation from ethanol to yield (14) (0.228 g, 46% yield) as pale yellow
powder: 1H NMR (300 MHz, CDCl3)
d
8.42 (t, J ¼ 1.8 Hz, 1 H), 8.35
(ddd, J ¼ 8.1, 1.8, 1.2 Hz, 1 H), 8.05 (d, J ¼ 13.5 Hz, 1 H), 7.87 (dt,
J ¼ 7.5, 1.2 Hz, 1 H), 7.68 (t, J ¼ 7.8 Hz, 1 H) 7.66 (d, J ¼ 13.8 Hz, 1 H).
E-pentafluoro(2-nitrovinyl)benzene (15). Prepared by the above
procedure (pentafluoro benzaldehyde, 0.538 g, 2.7 mmol). Purified
by filtration through a short plug (SiO2, 50% EtOAc in hexanes)
followed by flash chromatography (SiO2, 10% EtOAc in hexanes) to
yield (15) (0.142 g, 22% yield) as yellow oil: 1H NMR (300 MHz,
N-acryloyl-N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyr-
imidin-4-yl)acrylamide (19) as white solid (0.019 g, 0.051 mmol,
16.2% yield). 1H NMR (300 MHz, CDCl3):
d 8.94 (s, 1 H), 7.50-7.45 (m,
2 H), 7.40-7.35 (m, 3 H), 6.31 (dd, J ¼ 16.7, 2.3 Hz, 2 H), 6.22 (dd,
J ¼ 16.7, 9.3 Hz, 2 H), 5.63 (dd, J ¼ 9.3, 2.3 Hz, 2 H), 1.9 (s, 9 H). 13
C
NMR (75 MHz, CDCl3): d 167.2, 155.4, 154.0, 153.5, 142.7, 132.1, 131.0,
CDCl3)
(282 MHz, CDCl3)
159.7 (m, 2 F).
d
8.04 (d, J ¼ 13.8 Hz, 1 H), 7.82 (d, J ¼ 13.8 Hz, 1 H); 19F NMR
129.8, 129.1, 129.0, 128.9, 110.2, 61.4, 29.3. HRMS (EIþ) m/z calc’d for
d
135.8 (m, 2 F), 146.6 (tt, J ¼ 20.7, 4.6 Hz, 1 F),
C
21H21N5O2 [M]þ: 375.1695, found 375.1695.
E-2-bromo-1-fluoro-4-(2-nitrovinyl)benzene (16). Prepared by
the above procedure (3-bromo-4-fluorobenzaldehyde, 0.500 g,
2.5 mmol). Purified by filtration through a short plug (SiO2, 50%
EtOAc in hexanes) followed by recrystallization from ethanol to
yield (16) (0.311 g, 51% yield) as yellow microcrystals: 1H NMR
4.1.3.2. Synthesis of compound 20. Synthesis of compound 20 see
Scheme 4. To a THF (10 mL, 0.1 M) solution of phenyl pyr-
azolopyrimidine (17) (0.25 g, 0.94 mmol) was Et3N (0.17 mL,
1.2 mmol). Chloroacetic chloride (0.1 mL, 1.2 mmol) was added
dropwise. After 12 h at ambient temperature, the mixture was
quenched with H2O (10 mL) and extracted with CHCl3 (3x10 mL).
The combined organic layers were dried with MgSO4, filtered, and
concentrated. The crude oil was purified by silica gel column
chromatography (1:1 hexanes/ethyl acetate) to provide 20 as white
(300 MHz, CDCl3)
1 H), 7.51 (d, J ¼ 13.8 Hz, 1 H), 7.49 (m, 1 H), 7.21 (t, J ¼ 8.4 Hz, 1 H);
13C NMR (125 MHz, CDCl3)
d
7.91 (d, J ¼ 13.5 Hz, 1 H), 7.77 (dd, J ¼ 6.6, 2.4 Hz,
d
161.1 (d, J ¼ 255 Hz), 137.7 (d,
J ¼ 2.9 Hz), 136.4, 134.2, 129.9 (d, J ¼ 10.6 Hz), 127.8 (d, J ¼ 3.4 Hz),
117.6 (d, J ¼ 22.9 Hz), 110.6 (d, J ¼ 21.9 Hz); 19F NMR (282 MHz,
solid (0.18 g, 56% yield). 1H NMR (300 MHz, CDCl3):
d 8.65 (br s, 2 H),
CDCl3)
d
100.3 (dd, J ¼ 11.8, 7.1 Hz, 1 F).
7.69 (br s, 2 H), 7.56-7.42 (m, 3 H), 4.37 (s, 2 H), 1.84 (s, 9 H). Many of
the peaks in the 13C NMR spectrum were broadened by a dynamic
process that was not investigated further. Poor signal-to-noise
prevented acquisition of good 13C NMR spectral data. HRMS
(FABþ) m/z calc’d for C17H19ON5Cl [M þ H]: 344.1273, found
344.1270.
4.1.3. Synthesis of pyrazolopyrimidine analogues
Pyrazolopyrimidines 17, 21, and 26 were synthesized from the
corresponding acid chloride [47,58] and Scheme 2 shows synthetic
Route of Pyrazolopyrimidine analogues.
4.1.3.1. Synthesis of compounds 18 and 19. Representative Procedure
for Acylation of Pyrazolopyrimidines see Scheme 3. To a solution of 4-
amino-1-t-butyl-3-phenylpyrazolo[3,4-d] pyrimidine (17) (0.103 g,
0.387 mmol) in CH2Cl2 (4 mL, 0.1 M) was added Et3N (0.043 mL,
0.31 mmol). The solution was cooled in an ice water bath, and
acryloyl chloride (0.027 mL, 0.314 mmol) was added dropwise.
After stirring for 30 min while warming to room temperature, the
solution was partially concentrated and purified directly by silica
gel column chromatography (10% ethyl acetate/hexanes). In addi-
tion to recovered starting material (0.0306 g, 0.1145 mmol, 29.6%
yield), three products were isolated:
4.1.3.3. Synthesis of compounds 22 and 23. Synthesis of compounds
22 and 23 see Scheme 5. 2-Naphthyl pyrazolopyrimidine (21)
(0.1134 g, 0.357 mmol) was acylated as described in the above
representative procedure using CH2Cl2 (3 mL), Et3N (0.1 mL,
0.72 mmol) and acryloyl chloride (0.05 mL, 0.547 mmol). Water
(10 mL) and NaHCO3 (10 mL, saturated aq.) were added and
extracted with CH2Cl2 (3 x 10 mL). The combined organic layers
were dried with Na2SO4, filtered, and concentrated under reduced
pressure. The crude yellow oil was purified by silica column chro-
matography (1:9->1:3->1:0 ethyl acetate/hexanes) to provide
three products in addition to recovered starting material (0.0184 g,
0.0466 mmol, 13% yield).
N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)
acrylamide (18) as white solid (0.015 g, 0.046 mmol,14.5% yield). 1H
N-(1-(tert-butyl)-3-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyr-
imidin-4-yl)acrylamide (22) as white solid (0.026 g, 0.069 mmol,
19% Yield). 1H NMR (300 MHz, DMSO)
d 11.23 (br s, 1 H), 8.82 (s,
1 H), 8.20 (s, 1 H), 7.91 (m, 3 H), 7.82 (d, J ¼ 8 Hz, 1 H), 7.50 (m, 2 H),
6.32 (dd, J ¼ 10, 17 Hz, 1 H), 5.73(d, J ¼ 17 Hz, 1 H), 5.51 (d, J ¼ 10 Hz,
1 H), 1.86 (s, 9 H). Many of the peaks in the 13C NMR spectrum were
Scheme 2. Synthetic Route of Pyrazolopyrimidine analogues.
Scheme 3. Synthesis of compounds 18 and 19.
10

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
0.16 mmol). The solution was cooled in an ice water bath and to it
added acryloyl chloride (0.012 mL, 0.14 mmol). The solution was
purified without workup by silica gel column chromatography (5%
ethyl acetate/benzene) to yield 24 as colorless oil (0.0015 g,
0.0039 mmol, 4.7% yield) along with recovered starting material
(0.017 g, 62.4%). 1H NMR (500 MHz, CDCl3):
d 8.88 (s, 1 H), 8.05 (d,
J ¼ 1.6 Hz,1 H), 7.91 (d, J ¼ 8.5 Hz,1 H), 7.86 (m, 2 H), 7.74 (dd, J ¼ 8.5,
1.8 Hz, 1 H), 7.52 (m, 2 H), 6.20 (dd, J ¼ 10.1, 1.9 Hz, 1 H), 6.10 (dd,
J ¼ 16.7,1.9 Hz,1 H), 5.46 (dd, J ¼ 16.7,10.1 Hz,1 H), 3.20 (s, 3 H),1.93
(s, 9 H). 13C NMR (125 MHz, CDCl3):
d 166.4, 157.7, 155.9, 153.8,
Scheme 4. Synthesis of compound 20.
142.6, 133.5, 133.5, 130.0, 128.8, 128.8, 128.7, 128.7, 128.3, 128.0,
127.0, 126.9, 125.9, 108.4, 61.7, 36.3, 29.4. HRMS (FABþ) m/z calc’d
for C23H24N5O [M þ H]: 386.1975; found 386.1969.
4.1.3.5. Synthesis of compound 27 and 28. Synthesis of compounds
27 and 28 see Scheme 7. Biphenyl pyrazolopyrimidine (26) (0.2 g,
0.58 mmol) was acylated as described in the above representative
procedure using CH2Cl2 (10 mL, 0.06 M), Et3N (0.104 mL,
0.75 mmol), and acryloyl chloride (0.04 mL, 0.44 mmol). Purifica-
tion by silica gel column chromatography (1:3 ethyl acetate/hex-
anes) yielded two products in addition to recovered starting
material (0.042 g, 0.12 mmol, 21% yield). Data for starting pyr-
Scheme 5. Synthesis of compounds 22 and 23.
azolopyrimidine (26): 1H NMR (300 MHz, CDCl3):
7.77 (m, 4 H), 7.65 (m, 2 H), 7.48 (m, 2 H), 7.39 (m, 1 H), 5.68 (br s,
2 H), 1.85 (s, 9 H). 13C NMR (125 MHz, CDCl3):
158.0, 154.7, 154.5,
d 8.37 (s, 1 H),
broadened by a dynamic process that was not investigated further.
Poor signal-to-noise prevented acquisition of good 13C NMR spec-
tral data. HRMS (FABþ) m/z calc’d for C22H22N5O [M þ H]: 372.1819;
found 372.1811.
d
141.9, 141.8, 140.6, 132.8, 129.2, 129.1, 128.2, 127.9, 127.3, 100.0, 60.7,
29.4. HRMS (FABþ) m/z calc’d for C21H22N5 [M þ H]: 344.1870;
found 344.1866.
N-acryloyl-N-(1-(tert-butyl)-3-(naphthalen-2-yl)-1H-pyrazolo
[3,4-d]pyrimidin-4-yl)acrylamide (23) as white solid (0.066 g,
N-(3-([1,10-biphenyl]-4-yl)-1-(tert-butyl)-1H-pyrazolo[3,4-d]
pyrimidin-4-yl)acrylamide (27) as white solid (0.022 g,
0.16 mmol, 43% Yield). 1H NMR (500 MHz, CDCl3)
d 8.94 (s,1 H), 7.92
(s, 1 H), 7.84 (d, J ¼ 8.4 Hz, 1 H), 7.81 (m, 1 H), 7.76 (m, 1 H), 7.61 (dd,
0.055 mmol, 9.5% yield). 1H NMR (300 MHz, DMSO‑d6):
d 11.10 (br s,
J ¼ 8.4, 1.2 Hz, 1 H), 7.48 (m, 2 H), 6.18 (ABX quartet, 4 H), 5.46 (dd,
1 H), 8.80 (s, 1 H), 7.68 (br m, 6 H), 7.48 (m, 2 H), 7.37 (m, 1 H), 6.4
(dd, J ¼ 17.1, 10.3 Hz, 1 H), 5.89 (d, J ¼ 17.1 Hz, 1 H), 5.66 (dd, J ¼ 10.3,
1.6 Hz, 1 H), 1.83 (s, 9 H). Many of the peaks in the 13C NMR spec-
trum were broadened by a dynamic process that was not investi-
gated further. Poor signal-to-noise prevented acquisition of good
13C NMR spectral data. HRMS (FABþ) m/z calc’d. for C24H24N5O
[M þ H]: 398.1975; found 398.1962.
J ¼ 9.1, 2.8 Hz, 2 H), 1.9 (s, 9 H). 13C NMR (125 MHz, CDCl3)
d 167.3,
155.6,154.1, 153.6,142.7,133.5, 133.4, 131.1,129.7, 129.5,128.8, 128.7,
128.4, 127.9, 126.9, 126.8, 126.4, 110.3, 61.8, 29.5. HRMS (FABþ) m/z
calc’d for C25H24N5O2 [M þ H]: 426.1925; found 426.1930.
4.1.3.4. Synthesis of compounds 24 and 25 [59]. Synthesis of com-
pound 24 and 25 see Scheme 6. To solution of NaH (0.0055 g,
0.1375 mmol, 1.4 equiv.) and pyrazolopyrimidine 21 (0.0277 g,
0.1 mmol) in THF (3 mL, 0.03 M) was added CH3I (0.006 mL,
0.096 mmol). The solution was heated to 60 ꢁC for 5 h. The solution
was purified without workup by silica gel column chromatography
(5% ethyl acetate/hexanes) to yield the monomethylated pyr-
azolopyrimidine (25) as white solid (0.0277 g, 0.0836 mmol, 87%
N-(3-([1,10-biphenyl]-4-yl)-1-(tert-butyl)-1H-pyrazolo[3,4-d]
pyrimidin-4-yl)-N-acryloylacrylamide (28) as white solid (0.030 g,
0.066 mmol, 11.5% yield). 1H NMR (300 MHz, CDCl3)
d 8.95 (s, 1 H),
7.54e7.64 (m, 6 H), 7.47 (m, 2 H), 7.38 (m, 1 H), 6.3 (ABX quartet,
JAB ¼ 16.6 Hz, JAX ¼ 3.2 Hz, JBX ¼ 8.5 Hz, 4 H), 5.63 (dd, J ¼ 8.5, 3.2 Hz,
2 H), 1.91 (s, 9 H). 13C NMR (125 MHz, CDCl3)
d 167.23, 155.4, 154.0,
153.5, 142.4, 141.9, 140.7, 131.0, 131.0, 129.8, 129.5, 129.1, 127.8, 127.6,
127.3, 110.1, 61.8, 29.5.
yield). 1H NMR (500 MHz, CDCl3):
d 8.47 (s,1 H), 8.11 (s,1 H), 8.01 (d,
J ¼ 8.4 Hz, 1 H), 7.93 (m, 2 H), 7.78 (dd, J ¼ 8.4, 1.6 Hz, 1 H), 7.56 (m,
2 H), 5.36 (br s, 1 H), 3.05 (d, J ¼ 4.9 Hz, 3 H), 1.86 (s, 9 H). 13C NMR
4.2. Protein purification
(125 MHz, CDCl3):
d 158.0, 154.8, 153.9, 141.7, 133.7, 133.4, 131.7,
129.4, 128.4, 128.1, 128.0, 127.0, 126.9, 126.4, 100.4, 60.6, 29.4, 28.0.
HRMS (FABþ) m/z calc’d for C20H22N5 [M þ H]: 332.1870; found
332.1885.
Plasmids and primers used in this study are listed in Table S1.
Proteins were purified as previously described [21,41]. E. coli BL21
(DE3) containing the plasmid was grown in LB medium containing
To a solution of methylated pyrazolopyrimidine 25 (0.0277 g,
0.0836 mmol) in CH2Cl2 (5 mL, 0.02 M) was added Et3N (0.022 mL,
Scheme 6. Synthesis of compounds 24 and 25.
Scheme 7. Synthesis of compound 27 and 28.
11

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
50
m
g/L ampicillin, which was shaken at 37 ꢁC to an OD600 of 0.5.
4.5. Human 26S proteasome activity assay
The cell culture was cooled down to 22 ꢁC and induced with 1 mM
isopropyl-beta-D-thiogalactopyranoside (IPTG) and harvested
8e10 h later by centrifugation. The cell pellet (z6 g from 2 L) was
suspended in the 30 mL lysis buffer [100 mM Tris (pH 7.4), 500 mM
Human 26S proteasome complex was affinity purified from
HEK293 cells that stably express tagged human Rpn 11, as described
[50]. Purified human 26S proteasome (19 nM) was incubated with
KCl, 5 mM MgCl2, 20 mM imidazole, 5% glycerol, 2 mM
b
-mercap-
5, 25, or 50
7.4), 20 mM MgCl2, 1 mM EDTA, 0.5 mM TCEP, and 100
30 min at room temperature and the fluorogenic proteasome
substrate (succinyl-Leu-Leu-Val-Tyr-AMC, 60 M; Boston Biochem)
was added to initiate the reaction. Fluorescence intensity was
monitored every 3 min over 30 min. IC50 values of the compounds
were calculated from sextuplicate using GraphPad Prism 7.0.
m
M of MG132 or PPA in assay buffer [50 mM Tris (pH
toethanol, and protease inhibitor tablet (Roche)]. The cells (held in
an ice bath) were lysed by six 30-s pulses of sonication, separated
by 2-min intervals. The lysate was centrifuged at 20,000ꢂg for
45 min at 4 ꢁC, and the resulting supernatant was loaded onto a Ni-
NTA column [5-mL suspension, preequilibrated with wash buffer
(50 mL, 50 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, and
20 mM imidazole)] and incubated at 4 ꢁC with rotation for 30 min.
The column was then flushed with wash buffer (100 mL), and His6-
tagged p97 was eluted by stepwise application of 10 mL of imid-
azole elution buffer (50 mM,100 mM,150 mM, 200 mM, or 250 mM
imidazole in wash buffer). Fractions from the 200-mM and 250-
mM imidazole steps were combined and concentrated with an
Amicon Ultra-15 centrifugal filter unit (nominal molecular weight
limit ¼ 100 kDa). The mixture (0.5 mL of 20 mg/mL) was then
fractionated with a gel filtration column (Tricorn Superdex 200; GE
Healthcare), eluted with GF buffer [20 mM HEPES (pH 7.4), 250 mM
KCl, and 1 mM MgCl2] at 0.5 mL/min flow rate, and fractions cor-
responding to an apparent molecular weight of 500e600 kDa were
collected and analyzed by 4e12% SDS/PAGE to evaluate purity
(Invitrogen). Fractions that contained p97 of ꢃ95% purity were
concentrated to 5 mg/mL, exchanged into storage buffer [20 mM
HEPES (pH 7.4), 250 mM KCl, 1 mM MgCl2, 5% glycerol, and 1 mM
DTT], aliquoted, frozen in liquid nitrogen, and stored at ꢀ80 ꢁC.
mM ATP] for
m
4.6. Western blot
TCR
were treated with 4
in the presence of 50
or MG132 (as a positive control) for 60 min or 120 min prior to
harvest. Samples were immunoblotted to detect p97, and TCR
a
-GFP (
a
-chain of the T-cell receptor fused to GFP) 293 cells
M MG132 for 1 h, washed, and then incubated
M cycloheximide plus 10 M test compound
m
m
m
a-
GFP. The level of p97 served as a loading control. Primary antibodies
used were anti-p97 (MA3-004, Thermo Scientific), and anti-GFP
(Y1030, 2BScientific), ECL reagent (WBKLS0500, MilliporeSigma)
and ChemiDoc MP Imaging System (Bio-Rad) was used to image the
blots.
4.7. Docking study
The structure of PPA was prepared using Chem3D by Cam-
bridgeSoft. The geometry and energy of the structure were opti-
mized using MM2 force field. AutoDock 4.2 was used to identify the
binding mode of PPA responsible for the activity. The receptor WT-
p97 in PDB format (PDB code: 5FTK) was obtained from the RCSB
protein data bank (https://www.rcsb.org/), which was treated by
the removal of water and the addition of hydrogen. The docking
results were visualized using Ligplotþ [60] and PyMol (version 0.99;
Delano Scientific, San Carlos, CA, USA).
4.3. ATPase activity
The purified protein was assayed in 50
mL ATPase assay buffer
(50 mM Tris pH 7.4, 20 mM MgCl2, 1 mM EDTA, 0.5 mM TCEP, and
0.01% Triton X-100) containing 200
incubation at room temperature, 50
m
m
M ATP. After a 60 min-
L Biomol Green reagent
(Enzo Life Sciences) was added to stop the reaction, and the
absorbance at 635 nm was measured using a BioTek Synergy Neo 2
plate reader. The eight-dose titrations were performed of the
compound to the reaction to determine the IC50 values of the
compounds. The results were calculated from six replicates using
GraphPad Prism 7.0.
4.8. Anti-proliferative assay
Anti-proliferative activity was measured using the CellTiter
Glo® Luminescent Cell Viability Assay (Promega G7572) according
to the manufacturer’s procedure. RMPI1640 or DMEM containing
5% FBS and 1% Penicillin-Streptomycin was used as the cell viability
assay medium. To find the linear relationship between the relative
luminescence unit and the number of viable cells, a standard curve
4.4. UbG76V-GFP and ODD-Luc degradation
for each cell line was generated. Generally, 30 mL of cell suspension
was plated in 384-well plates (Greiner 781080) with serial 2-fold
dilutions (from 30000 to 284 cells per well). Twenty-four hours
This assay was described previously [41]. UbG76V-GFP/ODD-Luc
HeLa cells were seeded as 5000 cells per well per 30
plate (Greiner 781091). Clear DMEM containing 2.5% FBS, 1%
Glutamine, and 1% Penicillin-Streptomycin (Thermo Fisher) was
used as an assay medium. The 4 M of MG132 (50 L of 4 mM
MG132 into 50 mL pre-warmed clear DMEM) was prepared, added
into each well (25
L), and incubated at 37 ꢁC for 60 min. Each well
was washed with 100 L PBS twice using a Biotek MultiFlo Micro-
plate dispenser and 30 L CHX/clear DMEM solution (50 L of
50 mM cycloheximide into 50 mL pre-warmed clear DMEM) was
added. 10 L of compound was added and incubated for 120 min at
37 ꢁC. Then each well was washed with 100
L PBS, and 20 L of
mL in 384 well
L
-
after seeding, 8 mL of assay media containing 5% DMSO was added
into each well, and the plates were incubated for an additional
48 h at 37 ꢁC in a 5% CO2 incubator. To test the anti-proliferative
activity of p97 inhibitors, cells were seeded at 750 or 3000 cells
per well according to the linear range determined from the stan-
dard curve of each cell line. Twenty-four hours after seeding, cells
were treated with the compounds (three-fold dilution, eight con-
centrations). After 48 h of treatment, cell viability was measured by
CellTiter Glo, and IC50 values were calculated using the percentage
of growth of treated cells versus the DMSO control. The results were
analyzed using GraphPad Prism 7.0.
m
m
m
m
m
m
m
m
m
assay medium was dispensed per well. ImageXpress High Content
Confocal Screening System was used to read the GFP signal. Next,
10
mL D-luciferin (2.5 mg/mL in PBS) was added, shaken for 5 s, and
4.9. Proteomics
incubated at r.t. for 10 min. Synergy NEO multi-mode microplate
reader was used to read the Luciferase signal. Data was calculated
from quadruplicate to obtain the IC50 using GraphPad Prism 7.0.
HCT116 cells were treated with DMSO, 15
6 h, and pellets were harvested. The LC-MS samples were prepared
mM or 30 mM PPA for
12

G. Zhang, S. Li, F. Wang et al.
European Journal of Medicinal Chemistry 213 (2021) 113148
by following the instructions of Thermo EasyPep Mini MS Sample
Prep Kit (REF A4006) and peptide concentration was tested through
Pierce Quantitative Fluorometric Peptide Assay (cat# 23290).
The LC-MS/MS experiments were performed using an EASY-nLC
1000 (ThermoFisher Scientific, San Jose, CA) connected to an
Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scien-
Weissman for PYR-41, C. C. Wu for b-nitrostyrene analogues
(compounds 7, 9,10,11,12), Y. Ye at NIDDK/NIH for providing yeast
Cdc48 and hamster NSF plasmids. A.C.J. was supported by NIH
Grant F32GM082000; A.F.G.G thanks the Natural Sciences and
Engineering Research Council (NSERC) of Canada for a PGS D
scholarship; R.J.D. was an HHMI Investigator, and this work was
funded in part by HHMI; This work was funded in part by the NIH-
NINDS (R01NS102279) to T.F.C. and NIH-NIGMS (R01GM080269) to
B.M.S. We thank the anonymous reviewers for constructive
criticism.
tific, San Jose, CA). The sample (1
onto an Aurora UHPLC Column (25 cm ꢂ 75
25075C18A, Ion Opticks) and separated over 136 min at a flow rate
of 0.35 L/min with the following gradient: 2e6% Solvent B
mg) in 0.1% FA solution was loaded
m
m, 1.6 m C18, AUR2-
m
m
(7.5 min), 6e25% B (82.5 min), 25e40% B (30 min), 40e98% B
(1 min), and 98% B (15 min). Solvent A consisted of 97.9% H2O, 2%
ACN, and 0.1% formic acid, and solvent B consisted of 19.9% H2O,
80% ACN, and 0.1% formic acid. An MS1 scan was acquired in the
Orbitrap at 120,000 resolution with a scan range of 350e1500 m/z.
The AGC target was 4 ꢂ 105, and the maximum injection time was
50 min. Dynamic exclusion was set to exclude features after 1 time
for 60 s with a 10-ppm mass tolerance. Higher-energy collisional
dissociation (HCD) fragmentation was performed with 35% colli-
sion energy after quadruple isolation of features using a 1.6 m/z
isolation window, 5 ꢂ 104 AGC target, and 35 ms maximum in-
jection time. MS2 scans were then also acquired by the Orbitrap
with 50,000 resolution. Ion source settings were as follows: ion
source type, NSI; spray voltage, 2400 V; ion transfer tube temper-
ature, 275 ꢁC. System control and data collection were performed
by Xcalibur software.
The proteomic analysis was performed through Proteome
Discoverer 2.4 (Thermo Scientific) using the Uniprot human data-
base and the Byonic search algorithm (Protein Metrics). Percolator
FDRs were set at 0.001 (strict) and 0.01 (relaxed). Peptide FDRs
were set at 0.001 (strict) and 0.01 (relaxed), with medium confi-
dence and a minimum peptide length of 6. Normalization was
performed on the total peptide amount. The limma analysis was
performed using R studio following the user guide [61]. The vol-
cano figures were plotted by GraphPad Prism 7.0. The Venn diagram
was performed using FunRich 3.1. The heatmap figure was plotted
by heatmapper (http://www.heatmapper.ca/). The gene ontology
analysis was performed by DAVID 6.8 (https://david-d.ncifcrf.gov/)
[62]. The enrichment dot bubble figure was plotted by http://www.
bioinformatics.com.cn. And the protein-protein interaction analysis
was performed using the STRING database (http://www.stringdb.
org/) [63].
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113148.
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Gang Zhang wrote the manuscript and analyzed the potential
interaction between PPA and p97 by molecular docking. Shan Li and
Feng Wang performed the anti-proliferative assay and proteomics
study. Amanda C. Jones, Alexander F. G. Goldberg, and Scott Virgil
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Declaration of competing interest
The authors declare that they have no known competing
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