Small molecule inhibition of the ubiquitin-specific protease USP2 accelerates cyclin D1 degradation and leads to cell cycle arrest in colorectal cancer and mantle cell lymphoma models

Article abstract of DOI:10.1074/jbc.M116.738567

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC₅₀ of 1.1 μM in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.

Full text of DOI:10.1074/jbc.M116.738567

JBC Papers in Press. Published on September 28, 2016 as Manuscript M116.738567
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.738567
USP2 inhibition impacts cyclin D1 and cell cycle
Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates Cyclin D1 Degradation
and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models
Mindy I. Davisa,b, Rajan Pragania,b, Jennifer T. Foxa, Min Shena, Kalindi Parmarc, Emily F.
Gaudianoc, Li Liua, Cordelle Tanegaa, Lauren McGeea, Matthew D. Halla, Crystal McKnighta, Paul
Shinna, Henrike Nelsona, Debasish Chattopadhyayd, Alan D. D’Andreac, Douglas S. Aulda,e, Larry
J. DeLucasd, Zhuyin Lia, Matthew B. Boxera,*, Anton Simeonova,*
a NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National
Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20892, USA. b These authors contributed
equally to the work. c Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical
School, Boston, MA, 02215, USA d University of Alabama at Birmingham, Birmingham, AL 35294,
USA. e Current address: Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research,
Cambridge, MA 02139, USA.
Running title: USP2 inhibition impacts Cyclin D1 and cell cycle
* To whom correspondence should be addressed: Anton Simeonov and Matthew B. Boxer, NIH/NCATS,
9800 Medical Center Drive, Rockville, Maryland, 20850, Telephone: (301) 217-4021; FAX: (301) 217-
5736; email: asimeono@mail.nih.gov and boxerm@mail.nih.gov.
Keywords: cancer biology, cell cycle, enzyme inhibitor, cyclin D1, drug discovery, ubiquitination, USP2,
ubiquitin-specific protease
ABSTRACT
Deubiquitinases
DNA repair. These effects by a small molecule
inhibitor support a key role for USP2 as a
regulator of cell cycle, DNA repair and tumor
cell growth.
are
important
components of the protein degradation
regulatory network. We report the discovery of
ML364, a small molecule inhibitor of the
deubiquitinase USP2 and its use to interrogate
the biology of USP2 and its putative substrate
Cyclin D1. ML364 has an IC50 of 1.1 µM in a
biochemical assay using an internally quenched
fluorescent di-ubiquitin substrate. Direct binding
of ML364 to USP2 was demonstrated using
microscale thermophoresis. ML364 induced an
increase in cellular Cyclin D1 degradation and
caused cell-cycle arrest as shown in western
blots and flow cytometry assays utilizing both
mino and HCT116 cancer cell lines. ML364,
and not the inactive analog 2, was
antiproliferative in cancer cell lines. Consistent
with the role of Cyclin D1 in DNA damage
response, ML364 also caused a decrease in
homologous recombination (HR)-mediated
The ubiquitination status of proteins can
affect their degradation by the proteasome, as
well as their conformation and subcellular
localization, thereby modulating diverse
processes including cell cycle and apoptosis.
This status is tightly regulated at both the
ubiquitination level by the E1, E2, E3 ubiquitin
ligase system and the deubiquitination level by
over 90 deubiquitinases (DUBs) (1). The largest
of the five DUB families is the ubiquitin-specific
proteases (USPs). USP2 is a DUB that removes
ubiquitin thereby stabilizing
a
range of
substrates including MDM2 (a regulator of p53)
(2), fatty acid synthase (3), Cyclin D1 (4) and
proteins important for the circadian rhythm, such
1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

USP2 inhibition impacts cyclin D1 and cell cycle
as Cry1 (5). Typically, ubiquitinated proteins
with K48-linkages are directed to the
proteasome for degradation, while K63-linked
ubiquitinated proteins can regulate more diverse
functions, such as innate immunity, localization
and activation (6,7). USP2 has been shown to
catalyze the hydrolysis of both linkage types (8).
USP2 is upregulated in various cancers
including prostate cancer and ovarian
carcinoma, which would be expected to lead to
an increase in the levels of deubiquitinated
variants of its corresponding substrates (3), and
has also been described as an oncogene due to
its ability to transform cells and provide
resistance to apoptosis (9). The USP2 substrate
fatty acid synthase is upregulated in breast,
prostate and ovarian cancers and is associated
with a poor prognosis (10-12). The best-
characterized substrate of USP2, Cyclin D1, is
important for cell cycle progression from G1 to
S in many cell types and is activated or
overexpressed in several cancers including
mantle cell lymphoma (13), a sub-type of non-
Hodgkin’s lymphoma, in which the disease is
often quite advanced by the time of diagnosis.
The most common genetic alteration in this
cancer is a t(11:14) chromosomal translocation
that leads to overexpression of Cyclin D1
(14,15). In 2009, Shan et al. reported that the
knockdown of USP2 arrests cancer cell growth
suggest that ML364 acts on USP2 and can be
used to interrogate the effect on USP2 substrates
in a cellular context.
RESULTS
ML364 Reversibly Inhibits USP2 in a
Biochemical Assay and Its Selectivity is
Assessed- A high-throughput screen resulted in
the identification of a sulfamido benzamide
chemical series that inhibited USP2 biochemical
activity. Further optimization through medicinal
chemistry, led to development of the active
compound ML364 and a structurally related
inactive counterpart compound 2 (Fig. 1A).
Using internally quenched fluorescent di-
ubiquitin substrates (Di-Ub IQF; Fig. 1B),
ML364 was determined to have an IC50 of 1.1
µM for the K48-linked substrate and 1.7 µM for
the K63-linked substrate, while 2 was inactive.
The compounds did not have an effect on the
detection system alone. The interaction of
ML364 with USP2 was reversible as determined
by measuring the recovery of enzyme activity
following a large dilution of the USP2-ML364
complex (Fig. 1G). ML364 was also tested in
other protease assays to assess selectivity (Table
1 and Fig. 1C): it was inactive against Caspase
6, Caspase 7, MMP1, MMP9, and USP15, but
did inhibit USP8 with an IC50 of 0.95 µM, in
agreement with the similarity between the active
sites of these two isozymes. In a panel of 102
kinases that included regulators the cell cycle
there was no binding observed to any of the 102
enzymes tested using 10 µM of ML364. (Table
2 and Fig. 2).
by
promoting
the
proteasome-mediated
degradation of Cyclin D1, thus raising the
possibility that small molecules specifically
targeting this deubiquitinase could be effective
chemotherapeutic agents for cancers addicted to
Cyclin D1 expression (4). A crystal structure of
USP2 and kinetic analysis of its interaction with
ubiquitin have been reported (16,17); however
only a few USP2 inhibitors have been described
and several of these bind covalently and/or are
nonselective (18-20). Herein we report the
ADME Characterization of ML364-
Preliminary ADME properties were determined
and while ML364 had low solubility in PBS 7.4
buffer (<2 µM), its solubility was more than 10-
fold higher in the assay buffer (28.9 µM; 20 mM
Tris pH 8, 2 mM β-mercaptoethanol, 0.05%
CHAPS), where it was stable for more than 48
hours (Table 3a). The logD at pH 7.4 (a measure
of lipophilicity) was 2.31 and the PAMPA
permeability was 82.2 x 10-6 cm/s, indicating
that the compound has properties that should
allow for cell membrane permeation. While the
stability in rat microsomes was modest (t1/2 = 15
identification of
a small molecule USP2
inhibitor, ML364, demonstrate its activity in
USP2 biochemical assays, and profile its
selectivity across a panel of proteases and
kinases. We also characterize the binding of
ML364 to USP2 and test its effects on cell
viability and the levels of Cyclin D1. Our results
2

USP2 inhibition impacts cyclin D1 and cell cycle
minutes), the stability in mouse (61% remaining
at 30 minutes) and human (84% remaining at 30
minutes) microsomes and in mouse plasma
(100% remaining at 30 minutes) were quite good
(Table 3b).
the cell-based and biochemical assays are in
concert with the expected cell permeability of
the inhibitor and support the notion that ML364
engages USP2 in the cell. ML364-mediated
Cyclin D1 destabilization was not due to a
reduction in USP2 protein levels (Fig. 3A), and
was proteasome dependent, as the addition of
the proteasome inhibitor MG132 restored Cyclin
D1 protein levels to those observed in the
DMSO control (Fig. 3C). To test whether
ML364 promoted Cyclin D1 degradation in a
cell line that relies on this protein for
transformation, the above experiments were
repeated using the mantle cell lymphoma cell
line, Mino. Again, ML364, but not compound 2,
reduced Cyclin D1 protein levels in a time-,
dose-, and proteasome-dependent manner (Fig.
3, D-F).
Binding
of
ML364
to
USP2
Demonstrated by Microscale Thermophoresis-
Using label-free microscale thermophoresis, a
method to measure binding affinities by
monitoring differential movement of particles in
a microscopic temperature gradient (21), the
interactions of ML364 and 2 with USP2 were
examined. ML364 bound USP2 with a Kd of 5.2
µM while inactive analog 2 did not bind (Fig. 1,
D-E); control experiments with USP2 alone
confirmed that the thermophoresis signature was
due to binding of active compound to the
enzyme (Fig. 1F). USP2 is fluorescent due to the
presence of several tryptophan residues, two of
which line the ubiquitin binding pocket (PDB
ID: 2NHE). Upon addition of ML364 there was
a dose-dependent decrease in USP2 tryptophan
fluorescence yield (Fig. 1D). To determine that
the observed binding interaction was not due to
quenching of tryptophan fluorescence but rather
to thermophoresis that probes the size, charge,
and solution entropy of molecules, dilutions of
USP2 alone were tested and the dose response
curve produced was in the opposite direction
indicating that the curve observed for USP2 in
the presence of dilutions of ML364 was not due
to the tryptophan quenching.
To directly identify the changes in
Cyclin D1 protein stability by ML364, we
expressed a Cyclin D1-firefly fusion protein in
cells as such fusion proteins are usually directly
targeted by ubiquitin ligases (22,23). Such an
approach is used to identify changes in protein
stability in cells exposed to Lenalidomide (22).
We used a plasmid encoding Cyclin D1 open
reading frame (ORF) fused to firefly luciferase
(FLuc) reporter and Renilla luciferase (RLuc)
reporter for normalization purpose as described
(22). When 293T cells were transfected with this
plasmid, the cells expressed Cyclin D1-FLuc
fusion protein as detected by western blotting
with antibodies against FLuc and Cyclin D1
(Fig. 4, B-C). We next transfected HCT116 cells
with this plasmid, treated them with inactive
compound or ML364, and measured Fluc as
well as RLuc enzyme activity. As expected, a
decrease in relative FLuc/RLuc activity was
observed in cells exposed to ML364 suggesting
a downregulation of Cyclin D1 fusion protein
(Fig. 4D). As a positive control, exposure of
cells to BIIB021 (a HSP90 inhibitor) also
resulted in decrease in relative FLuc/RLuc
activity (Fig. 4E). Reduced FLuc/RLuc activity
was also observed in 293T cells exposed to
ML364 (Fig. 4F). Collectively, these results
suggest that ML364 directly targets Cyclin D1
for degradation via USP2 inhibition.
Effect of USP2 Inhibition by ML364 in
Cells Investigated by Western Blotting- To
determine the effect of USP2 inhibition on
Cyclin D1 stability, HCT116 colorectal cancer
cells were treated with ML364 and Cyclin D1
protein levels were monitored by both western
blotting (Fig. 3, A-C) and in-cell ELISA (Fig.
4A). Whereas Cyclin D1 protein level remained
unchanged in cells treated with the inactive
compound 2, it decreased in both a time- and
dose-dependent manner following treatment
with ML364. Quantification of the western blot
results revealed an IC50 value of 0.97 µM, which
is consistent with the results from the
biochemical assays discussed above (Fig. 1 and
Table 1). The similar potencies for ML364 in
3

USP2 inhibition impacts cyclin D1 and cell cycle
Effect of USP2 Inhibition by ML364 in
Cells Investigated by Flow Cytometry- Since
Cyclin D1 regulates the G1-to-S phase transition
of the cell cycle, the cell cycle profiles of
ML364-treated HCT116 and Mino cells were
determined by both flow cytometry (Fig. 5, A-B)
and high content imaging (IC50 = 2 µM; Fig.
5C): in contrast to cells treated with 2, which
were able to progress to the G2/M phase of the
cell cycle, both cell types remained largely in G1
phase when treated with ML364. It is important
to note that for these assays, cells were co-dosed
with nocodazole, which blocks cells in G2/M
phase, to increase the ability to visualize the
changes in the G1 levels. Consistent with
ML364-induced cell cycle arrest, clonogenic
assays revealed that the growth of HCT116 cells
was severely inhibited following chronic
treatment with ML364 (Fig. 5, D-E, IC50 = 3.6
µM), whereas cells treated with 2 proliferated at
a rate comparable to the DMSO-treated control
(Fig. 5, F-G). Assessment of cell viability in
HCT116 cells and Mino cells using cellular ATP
content as the readout also showed that ML364
decreased cell viability (Fig. 6A). In addition to
HCT116 and Mino cells, LnCAP cells (prostate
cancer cells) and MCF7 cells (breast cancer
cells) also showed decrease in cell viability upon
exposure to ML364 in a dose dependent manner
(Fig. 6, B-C). The decrease in cell viability in
these cell lines correlated with Cyclin D1
degradation (Fig. 6D). We next determined
whether additional targets of USP2, namely,
MDM2 and FAS, are affected by ML364.
Interestingly, FAS but not MDM2 protein levels
were moderately reduced after ML364 exposure
in LnCAP prostate cancer cells (Fig. 6E,F).
cell-based DR-GFP reporter assay to measure
the activity of HR-mediated DNA repair.
Interestingly, ML364 caused a decrease in HR-
mediated DNA repair in DR-GFP U2OS cells
(Fig. 7A). We next analyzed RAD51 foci, a
surrogate marker for efficient HR-mediated
DNA repair, after inhibition of USP2 by
ML364. Consistent with the results from DR-
GFP reporter assay, ML364 exposure in Hela
cells caused a decrease in IR-induced RAD51
foci (Fig. 7, B-C). Together, these data indicate
that inhibition of USP2 by ML364 causes a
decrease in HR suggesting the role of USP2 in
DNA repair.
DISCUSSION
Bortezomib and carfilzomib are
clinically used to treat multiple myeloma (both)
and mantle cell lymphoma (bortezomib only)
(25,26). While these drugs inhibit the
proteasome in both normal and cancer cells,
herein we sought to impact one enzyme (USP2)
that controls the fate and levels of several
oncogenic substrates by specifically promoting
their degradation. With the DUB activity of
USP2 inhibited, these oncogenic proteins are
expected to remain ubiquitinated and targeted
for proteasomal degradation as shown herein for
Cyclin D1, while leaving the proteasome
functionally intact. Shan et al. had previously
tested 76 DUBs and found that only USP2 was
able to deubiquitinate Cyclin D1 in human cells
(4). Importantly, knockdown of USP2 was
shown previously to induce growth arrest in
cells that depend on Cyclin D1 for cell growth
but to have minimal effects on normal
fibroblasts and no effect on cancer cell types that
do not express Cyclin D1, such as SAOS2 (4).
Using both HCT116 cells, used in the
previous siRNA work by Shan et al. (4), and the
Mino mantle cell lymphoma line, noted for its
very high Cyclin D1 levels (Broad-Novartis
Cancer Cell Line Encyclopedia), ML364 was
shown to impact the levels of Cyclin D1 through
an increase in proteasome-mediated degradation.
Interestingly, the IC50 observed in the mantle
cell lymphoma model was right-shifted relative
to that observed in HCT116, which could be a
Effect
of
USP2
inhibition
on
homologous recombination (HR)-mediated DNA
repair-Recent studies suggest that Cyclin D1
regulates DNA damage response (24). Cyclin
D1 interacts with RAD51, a critical component
of homologous recombination (HR)-mediated
DNA repair. Reduction of Cyclin D1 levels in
cancer cells results in impaired HR-mediated
DNA repair and increased DNA damage (24).
We therefore tested whether inactivation of
USP2 by ML364 alters DNA repair. We used a
4

USP2 inhibition impacts cyclin D1 and cell cycle
consequence of the very high levels of Cyclin
D1 in those cells (14,15). Decrease in Cyclin D1
stability by ML364 was also confirmed by
expressing a Cyclin D1 luciferase fusion protein
in a reporter assay. ML364 affected cell cycle,
blocking the cells in G0/G1 phase and
preventing cell cycle progression as observed by
flow cytometry and high content imaging
(Figure 8). As mitotic arrest is poorly tolerated
by cancer cells, viability was decreased in
multiple cancer cell lines upon treatment with
ML364. Additionally, the size and number of
colonies in a 10-day colony forming assay were
reduced upon treatment with the inhibitor. While
ML364 displays similar potency against USP8
and USP2, USP8 is not known to be involved in
the cell cycle, but rather has a role in endosomal
trafficking (27). Indeed, USP8 has been
described as a novel target for overcoming
gefitinib-resistance in lung cancer indicating a
potential additional use for ML364 (28). Our
data suggest that besides regulating cell cycle,
USP2 also plays a role in HR-mediated DNA
repair by stabilizing Cyclin D1. Recent studies
suggest that Cyclin D1 regulates DNA damage
repair (24,29). Cyclin D1 facilitates HR-
mediated DNA repair through the recruitment of
RAD51 to double stranded breaks and Cyclin
D1 depletion causes a reduction in HR (24).
Therefore, promoting the degradation of Cyclin
D1 through the inhibition of USP2 using
ML364 presents a new avenue for treating
Cyclin D1 driven cancers. Indeed, using a DR-
GFP reporter assay and by assessing DNA
damage-induced RAD51 foci, we found that
ML364 causes HR defect. These data suggest
that USP2 may be a novel therapeutic target for
inhibiting HR activity in Cyclin D1 driven
cancers. Suppressing homologous recombination
using this strategy would enhance the effect of
genotoxins, such as PARP inhibitors, which
specifically target HR deficient tumors.
(19). ML364 is an extensively characterized
non-covalent USP2 inhibitor and as such is an
important probe of USP2 biology. Of the six
additional proteases tested, only the closely
related USP8 was inhibited, indicating that
ML364 is not a general protease inhibitor.
Further, 102 kinases were tested, many of which
are involved in the cell cycle, and ML364 did
not bind to any, thereby supporting USP2 as the
target leading to the observed cell cycle arrest
phenotype observed. While the ADME
properties of ML364 are suitable for the
biochemical and cell-based assays used here, an
improved molecule may be needed for future in
vivo testing. While in this initial study we
focused on Cyclin D1, there are additional
putative USP2 substrates that can be
interrogated in relevant cell lines using ML364.
It is envisioned that ML364 can be used in
proteomic studies to elucidate heretofore
unknown substrates of USP2. Our provision of
ML364 to the research community should spur
studies in these and other directions.
EXPERIMENTAL PROCEDURES
General Methods for Chemistry- All air-
or moisture-sensitive reactions were performed
under positive pressure of nitrogen with oven-
dried glassware. Anhydrous solvents such as
dichloromethane,
(DMF), acetonitrile, methanol and triethylamine
were purchased from Sigma-Aldrich.
N,N-dimethylformamide
Preparative purification was performed on a
Waters semi-preparative HPLC system. The
column used was a Phenomenex Luna C18 (5
micron, 30 x 75 mm) at a flow rate of 45
mL/min. The mobile phase consisted of
acetonitrile and water (each containing 0.1%
trifluoroacetic acid). A gradient of 10% to 50%
acetonitrile over 8 minutes was used during the
purification. Fraction collection was triggered by
UV detection (220 nM). Analytical analysis was
performed on an Agilent LC/MS (Agilent
Technologies, Santa Clara, CA). Purity analysis
was determined using a 7 minute gradient of 4%
to 100% acetonitrile (containing 0.025%
trifluoroacetic acid) in water (containing 0.05%
Previously reported USP2 inhibitors
include patented 2-cyano pyrimidines (potency
range 100 nM - 50 µM, likely reversible
covalent modifiers) (18), and chalcone
compounds (double-digit micromolar potency
but also shown to inhibit many other DUBs)
5

USP2 inhibition impacts cyclin D1 and cell cycle
trifluoroacetic acid) with an 8 minute run time at
a flow rate of 1 mL/min. A Phenomenex Luna
C18 column (3 micron, 3 x 75 mm) was used at
a temperature of 50 °C using an Agilent Diode
Array Detector. Mass determination was
performed using an Agilent 6130 mass
spectrometer with electrospray ionization in the
bicarbonate (2 x 30 mL), and saturated brine (2
x 30 mL). The resulting organic solution was
dried over MgSO4, and concentrated under
reduced vacuum. The crude oil was then purified
via reverse phase chromatography to furnish
ML364 as an off-white solid (219 mg, 31%). 1H
NMR (400 MHz, DMSO-d6) δ 12.83 (bs, 1H),
10.27 (bs, 1H), 8.05 – 7.88 (m, 3H), 7.76 (s,
1H), 7.67 – 7.55 (m, 3H), 7.45 – 7.42 (m, 3H),
7.36 (t, J = 7.3 Hz, 1H), 7.32 – 7.22 (m, 2H),
2.26 (s, 3H); 19F NMR (376 MHz, DMSO-d6) δ
–62.1; LC-MS Retention Time: t1 = 7.107 min;
HRMS (ESI) m/z (M+H)+ calcd. for
C24H18F3N3O3S2, 518.0814; found 518.0810.
3-(4-
methylphenylsulfonamido)isonicotinic acid (SI-
2)- A mixture of 3-aminoisonicotinic acid (181
mg, 1.311 mmol), sodium carbonate (347 mg,
3.28 mmol), and water (3.28 mL) was treated
with tosyl chloride (250 mg, 3.28 mmol) (Fig.
9B). This mixture was stirred at 80 ˚C for 2 hr.
After cooling to 23 ˚C, 1N HCl was added
dropwise (gas evolves from sodium carbonate
neutralization) until the mixture reaches pH 2.
The resulting precipitated solid was collected by
vacuum filtration, washed with water, and dried
by high vacuum overnight to afford intermediate
sulfonamide SI-2 as a pure off-white solid (60
mg, 16%).
1
positive mode. H NMR spectra were recorded
on Varian 400 MHz spectrometers. Chemical
shifts are reported in ppm with non-deuterated
solvent (DMSO-d5 peak at 2.50 ppm) as internal
standard for DMSO-d6 solutions. All of the
analogs tested in the biological assays have a
purity greater than 95% based on LCMS
analysis. High resolution mass spectrometry was
recorded on Agilent 6210 Time-of-Flight
LC/MS system. Confirmation of molecular
formulae was accomplished using electrospray
ionization in the positive mode with the Agilent
Masshunter software (version B.02).
2-(4-methylphenylsulfonamido)-4-
trifluoromethylbenzoic acid (SI-I)- A mixture of
2-amino-4-trifluoromethylbenzoic acid (1.08 g,
5.25 mmol), sodium carbonate (1.39 g, 13.11
mmol), and water (10.5 mL) was treated with
tosyl chloride (1.0 g, 5.25 mmol) (Fig. 9A). This
mixture was stirred at 80 ˚C for 2 hr. After
cooling to 23 ˚C, 1N HCl was added dropwise
(gas
evolves
from
sodium
carbonate
neutralization) until the mixture reached pH 2.
The resulting precipitated solid was collected by
vacuum filtration, washed with water, and dried
by high vacuum overnight to afford intermediate
sulfonamide SI-1 as a pure off-white solid (1.07
g, 57%).
3-(4-methylphenylsulfonamido)-N-
(pyridin-4-yl)isonicotinamide (2)- A mixture of
sulfonamide SI-2 (40 mg, 0.137 mmol), pyridin-
4-amine (12.9 mg, 0.137 mmol), DIPEA (96 µL,
0.547 mmol), and DMF (0.68 mL) was treated
with HATU (78 mg, 0.205 mmol). This mixture
was stirred at 80 ˚C for 12 hr. The resulting
solution was purified directly via reverse phase
chromatography to furnish the TFA salt of
compound 2 as an off-white solid (41 mg, 62%).
1H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J =
7.1 Hz, 2H), 8.54 (s, 1H), 8.28 (s, 1H), 8.03 (d, J
= 7.1 Hz, 2H), 7.66 (d, J = 5.1 Hz, 1H), 7.56 (d,
J = 8.2 Hz, 2H), 7.30 (d, J = 7.5 Hz, 2H), 2.31
(s, 3H); 19F NMR (376 MHz, DMSO-d6) δ –
74.2; LC-MS Retention Time: t1 = 1.527 min;
HRMS (ESI) m/z (M+H)+ calcd. for
C18H16N4O3S, 369.1016; found 369.1005.
2-(4-methylphenylsulfonamido)-N-(4-
phenylthiazol-2-yl)-4-
(trifluoromethyl)benzamide (ML364)- A mixture
of sulfonamide SI-1 (0.4 g, 1.11 mmol), 4-
phenylthiazol-2-amine (0.20 g, 1.11 mmol),
DIPEA (0.78 mL, 4.45 mmol), and DMF (2.78
mL) was treated with HATU (0.51 g, 1.34
mmol). This mixture was stirred at 80 ˚C for 2
hr. The resulting solution was quenched with
water, and the organic layer was extracted with
ethyl acetate (2 x 30 mL). The combined organic
layers were washed with saturated ammonium
chloride (2 x 30 mL), saturated sodium
6

USP2 inhibition impacts cyclin D1 and cell cycle
General Methods for Biology- The
catalytic core of USP2a (EC 3.4.19.12 and
control was dispensed into column 4. Using a
1536 pintool (Kalypsys Systems, San Diego,
CA, USA), 23 nL of compounds were then
added and the plate was incubated for 10
minutes. To initiate the reaction, 1 µL of 100
nM Di-Ub IQF substrate (Lifesensors, K48-4 or
K63-3) in 20 mM Tris pH 8, 2 mM β-
mercaptoethanol and 0.05% CHAPS was added
and the fluorescence signal (excitation 525 nm
and emission 598 nm, bodipy mirror and 0.5 sec
exposure) was monitored in kinetic mode on a
Viewlux high-throughput CCD imager (Perkin
Elmer, Waltham, MA, USA); the apparent
reaction rate was calculated using the signal
change during the first 15 minutes, which was
within the linear region of this assay.
Additionally, to verify that the compounds did
not interfere with the detection system, the effect
of compound on the substrate alone was tested
by omitting the enzyme from the above reaction.
Reversibility and Substrate Competition-
Following the method described in Copeland
(32), to test the reversibility of the ML364
binding to USP2, ML364 at a concentration ten
times its IC50 and 100x the concentration of
USP2 used in the K48-4 assay described above
were incubated for 1 hour at which point the
sample was diluted 100-fold in assay buffer. The
activity of this diluted sample was tested as
described above for the miniaturized K48-4 and
K63-3 enzyme assays.
UniProt
#
O75604) was obtained from
Lifesensors and Progenra (Malvern, PA, USA)
for the high-throughput screen. We produced
USP2 for additional studies as had been
previously reported in PDB ID 3NHE. Buffer
components were purchased from Sigma (St.
Louis, MO, USA) unless otherwise specified.
Experiments were run at room temperature
unless otherwise specified. Medium binding
black or white solid-bottom 1536-well plates
(assay plates) and 1536-well polypropylene
plates (compound plates) were purchased from
Greiner Bio One (Monroe, NC, USA).
Compound plating and data analysis-
Preparation of the compound library for
quantitative high-throughput screening (qHTS)
has been described previously (30). The library
was formatted into columns 5-48 of 1536-well
compound plates. A control plate was made in a
compound plate in columns 1-4 using a Cybi-
Well (CyBio, Jena, Germany) to transfer
solutions from a 384 compound storage plate to
the 1536 plate compound plate. Columns 1, 3,
and 4 contained DMSO and column 2 contained
2 M N-ethylmaleimide in dimethyl sulfoxide
(DMSO, certified ACS grade, Thermo-Fisher
Scientific (Pittsburg, PA, USA)). Data were
normalized to control columns representing
maximum signal (no enzyme or no cells) and
minimum signal (all components). Screening
data were corrected and normalized, and
concentration-response curves were derived
using in-house algorithms (31).
Selectivity Assays- Using Kinomescan
(DiscoveRx, Fremont, CA, USA), 102 kinases
were tested for inhibition by 10 µM ML364.
MMP1 and MMP9 data were obtained from
Reaction Biology (Malvern, PA, USA) using
their standard conditions (5 µM of the FRET
peptide). Caspase 6 and caspase 7 assays were
run using the Caspase 6 and Caspase 3/7 Glo
kits from Promega (Madison, WI, USA). This
kit couples the activity of the cysteine-aspartic
acid protease, i.e., the caspase, to luciferase.
First, 2.5 µL of Caspase 6 (0.5 U/mL; Enzo Life
Sciences, Farmingdale, NY, USA) of Caspase 7
(0.5 U/mL; Enzo Life Sciences) in 10 mM
Hepes pH 7.2, 2 mM DTT, 10% glycerol and
0.05% CHAPS was dispensed into a white 1536-
well plate. The substrate is at 5 µM in this assay,
Ub-Chop2
Screen-
This
assay
technology was described previously (20), and
details of the high-throughput screen and
counterscreen executed in 1536-well plate
format from which the chemical series discussed
herein was identified is publicly available at
PubMed ID: 463254 and 493169.
K48-4 and K63-2 Assay- Using a Flying
Reagent Dispenser (Beckman Coulter, Fullerton,
CA, USA), 2 µL of 10 nM USP2 in 20 mM Tris
pH 8, 2 mM β-mercaptoethanol and 0.05%
CHAPS was dispensed in a black 1536-well
plate in columns 1-3, 5-48 and a no enzyme
7

USP2 inhibition impacts cyclin D1 and cell cycle
which is the Km for the substrate per the
manufacturer. Then, 23 nL of compounds were
dispensed using the pintool and incubated for 30
minutes at room temperature. Lastly, 2.5 µL of
Caspase Glo reagent (either Caspase Glo 6 or
Caspase Glo 3/7) was added and the
luminescence was monitored kinetically using a
Viewlux for a total of 50 minutes (1 sec
exposure).
quenching by the compound could be
responsible for the observed IC50 curve.
Cell culture- HCT116 cells were
cultured in Dulbecco’s Minimum Eagle Medium
(DMEM) + GlutaMAX (Invitrogen, Carlsbad,
CA, USA) supplemented with 10% FBS
(Hyclone Laboratories, Waltham, MA, USA)
and 50 U/mL penicillin and 50 µg/mL
streptomycin
(Invitrogen).
Cells
were
Three µL of USP8core (140 nM,
maintained at 37 °C under a humidified
atmosphere and 5% CO2. Mino cells, a cell line
established and characterized as described in
(33) and LnCAP cells were cultured in RPMI-
1640 (Gibco, Carlsbad, CA, USA) with 10%
FBS and 50 U/mL penicillin and 50 µg/mL
streptomycin. MCF7 cells were grown in
DMEM with 10% FBS, 10 µg/ml insulin
(Sigma) and 50 U/mL penicillin and 50 µg/mL
streptomycin. U2OS-DRGFP cells and Hela
cells were cultured in DMEM with 10% FBS,
and 50 U/mL penicillin and 50 µg/mL
streptomycin.
Lifesensors) or USP15 (125 nM, Lifesensors)
were added to black 1536-well plates that had
been prespotted with either 23 nL of DMSO or
test compound using an acoustic dispenser
(ATS-100 acoustic dispenser, EDC Biosystems,
Freemont, CA, USA) to transfer the DMSO or
DMSO/compound from a Greiner 384-well
cyclic olefin copolymer source plate to the 1536-
well assay plate. Following incubation for 30
minutes, 1 µL of Di-Ub IQF K48-02 (100 nM)
was added and the reaction was monitored
kinetically using a Viewlux as for the USP2
assay above.
Western Blotting- Cells were grown in
Microscale Thermophoresis- Following
6-well tissue-culture plates and treated as
indicated. To obtain total lysate, the cells were
resuspended in lysis buffer [50 mM Tris, pH 7.5,
150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA,
1X protease inhibitor cocktail (Cell Signaling
Technology, Danvers, MA, USA)] and lysed on
ice for 30 min. Proteins were separated by SDS-
PAGE using a Novex 4–12% Tris-glycine gel
(Life Technologies, Grand Island, NY, USA)
and transferred to a nitrocellulose membrane
using the iBlot Dry Blotting System (Life
Technologies). The membrane was blocked with
5% nonfat dry milk in PBS/0.1% Tween 20 for 1
h at room temperature, incubated with primary
antibody in PBS/0.1% Tween 20 overnight at 4
°C, and then incubated with HRP-conjugated
secondary antibody in PBS/0.1% Tween 20 for 1
h at room temperature. After each incubation,
the membrane was washed with PBS/0.1%
Tween 20. Proteins were visualized using
SuperSignal West Dura Extended Duration
Substrate (Thermo Fisher, Waltham, MA, USA)
and the Chemidoc XRS Imaging System (Bio-
Rad, Hercules, CA, USA). The following
antibodies were used: mouse anti-Cyclin D1
the Manufacturer’s protocol, a 16-point titration
series of ML364 in DMSO (40 µM final, 1:2
dilutions), were transferred to the protein (1 µM)
in a buffer containing 20 mM Tris pH 8, 2 mM
β-mercaptoethanol and 0.05% CHAPS, samples
were loaded into Monolith NT hydrophilic-
treated Label-free capillaries (NanoTemper
Technologies, München, Germany). The buffer
alone and compound in buffer were tested and
found not to be fluorescent. A capillary scan was
performed followed by the successive
measurement of thermophoresis in each
capillary on a NanoTemper Monolith NT.115
LabelFree instrument at room-temperature (UV-
light emitting diode excitation and 350 nm
emission). The IR laser power and the LED
power conditions are described in the figure
captions. A laser on-time of 30 seconds and a
laser-off time of 5 seconds were used. The
experiment was performed in duplicate. Curve
fitting was performed using GraphPad Prism 5
(La Jolla, CA, USA). As a control, dilutions of
USP2 were also tested per the manufacturer’s
protocol to determine whether tryptophan
8

USP2 inhibition impacts cyclin D1 and cell cycle
(DCS6, #2926, Cell Signaling, 1:1,000 dilution),
rabbit anti-Cyclin D1 (#2922, Cell Signaling,
1:1,000 dilution), fatty acid synthase (FAS) (SC-
48537, Santacruz, 1:1000 dilution), MDM2 (Ab-
1, # OP46, Calbiochem, 1:500 dilution), rabbit
anti-USP2 (AP2131c-ev, Abgent, 1:1000
dilution), rabbit anti-GAPDH (ab9485, Abcam,
San Diego, CA, USA, 1:5,000 dilution), anti-
tubulin (sc-32293, Santacruz, 1:1,000 dilution),
HRP-conjugated anti-mouse IgG (#7076, Cell
terminated through the addition of an equal
volume of 1M HCl. Absorbance at 450 nm was
quantified on an Envision Multilabel Plate
Reader (Perkin Elmer, Waltham, MA, USA).
Cell cycle analysis- Cells were treated
with 10 µM compound and 100 nM Nocodazole
for 16 hours, harvested, and fixed with 70%
ethanol. The cells were then resuspended in 500
µl RNase/Propidium Iodide solution (Cell
Signaling Technology) supplemented with a
1:50 dilution of 1 mg/mL Propidium Iodide
(Life Technologies) and incubated at room
temperature for 15 minutes. Cell cycle analysis
was carried out by flow cytometry using the
Signaling,1:5,000
dilution),
and
HRP-
conjugated anti-rabbit IgG (#JM-6401-05, MBL
International Corporation, Woburn, MA,
USA,1:5,000 dilution. When necessary, the
membranes were stripped using Restore Western
Blot Stripping Buffer (Thermo Fisher). Band
intensities were quantified using ImageJ. The
ratio of Cyclin D1 or USP2 to GAPDH protein
levels at each compound concentration,
normalized against the start time or vehicle
controls, was used to determine the IC50. Cyclin
D1 and tubulin proteins in LnCAP cells and
MCF7 cells were assessed by western blotting,
as described (34).
IntelliCyt
HTFC
screening
system
(Albuquerque, NM, USA).
Cell Cycle Imaging Assay- HCT116
cells were seeded at a density of 600 cells/well
in a 96-well clear bottom black Costar plate and
incubated overnight at 37 °C and 5% CO2. The
media was removed and 50 µL of media (+/-
100 nM nocadazole) with 196 nL of compound
in DMSO was added to the cells. The
media/compound mix was created in a separate
96-well plate using an acoustic dispenser for the
compound followed by pipet addition of the
media. Cells were washed PBS, fixed with
formaldehyde (8%, Electron Microscopy
Sciences, Hatfield, PA) and permeabilized with
0.2% Triton X in PBS. This was removed and
25 µL of propidium iodide/RNAse (Cell
Signaling Technology) was added and the plate
was incubated for 1 hr at 37 °C. Plates were then
read on an Acumen eX3® microplate cytometer
(TTP LabTech Ltd., Melbourn, UK; excitation
488 nm, emission 575-640 band pass and 640
dichroic, PMT 500). The presence of active
USP2 leads to higher fluorescence due to cell
cycle progression from G0/G1 to S/M/G2.
In-Cell ELISA- HCT116 cells were
seeded into clear 96-well plates (Costar,
Corning, Tewksbury, MA, USA) at a density of
25,000 cells/well and treated with compounds
for 24 hours. The cells were then fixed with 4%
paraformaldehyde for 15 min, permeabilized
with PBS/0.1% Triton X-100 for 30 min, and
blocked with 2X Blocking Buffer (Sigma) for 2
hours at room temperature. After each step, the
cells were washed extensively with PBS using
the BioTek Elx406 Microplate Washer. To
quantify Cyclin D1 protein levels, the cells were
incubated with mouse anti-Cyclin D1 (DCS6,
Cell Signaling, 1:500 dilution) in 1X Blocking
Buffer (Sigma) overnight at 4°C, and then
incubated with HRP-conjugated anti-mouse IgG
(Cell Signaling,1:500 dilution) in 1X Blocking
Buffer (Sigma) for 2 hrs at room temperature.
After each incubation, the cells were washed
with PBS/0.1% Tween 20 using the BioTek
Elx406 Microplate Washer. 100 µL TMB Liquid
Substrate System for ELISA (Sigma) was then
added to each well, and after allowing the color
to develop for 5 minutes, the reaction was
Clonogenic Assay- 500 cells in 2 mL
media were incubated in a 6-well dish at 37 °C
for 2 days at which time 3 µL of compound (15,
5, 1.6, 0.56, 0.19 or 0 µM final concentration) in
DMSO was added and the cells were allowed to
grow for an additional 10 days. Then the media
is removed, the cells are washed with PBS and
crystal violet in 10% methanol was added to fix
and stain the cells. After a 30 min incubation,
9

USP2 inhibition impacts cyclin D1 and cell cycle
the crystal violet was removed and the plate was
rinsed with water and allowed to air dry
overnight. The cells are imaged using a Typhoon
FLA 9500 (GE Healthcare Life Sciences,
Pittsburg, PA, USA) in end-point mode using
Cy5 detection (Ex 635 and Em long pass red
filter) and the colony size and number was
quantitated using the Typhoon analysis software.
The total fluorescence per well was used to
derive the IC50values.
well plates (BD Biosciences) with 20,000
cells/100 µL per well. A transfection mixture
(20 µL per well) containing lipofectamine and
ORFeome Luciferase fusion plasmid DNA was
added on the day of transfection. 24 hrs after
transfection, cells were treated with DMSO or
compounds (20 µL per well). The firefly and
renilla luciferase signals were quantified using
the Dual-glo assay kit (Promega, # E2920)
according to manufacturer’s instructions at 24
hrs after the drug treatments.
Cell Viability Assay- Cells were seeded
at a density of 500 cells/5 µL well in a 1536-
well white solid bottom plate and incubated for
four hours at 37 °C and 5% CO2. Then, 23 nL of
compound solution in DMSO was added and the
plate was incubated for 72 hours. 3 µL Promega
CellTiter Glo reagent (Madison, WI, USA) was
then added to each well, and the plates were read
for luminescence using a Viewlux to determine
any compound effects on cell viability. For
experiments with LnCAP and MCF7 cells, cells
were seeded at a density of 2000 cells/100 µL
well in a flat bottom 96-well plate for 24 hrs at
37 °C and 5% CO2. Then DMSO or ML364 was
added and the plates were incubated for 24 or 48
hrs. 40 µL of CellTiter Glo reagent was added to
each well before reading the luminescence.
Homologous recombination (HR) analysis- HR
activity was analyzed by DR-GFP reporter assay
as previously described (34,35). U2OS-DRGFP
cells carrying a chromosomally integrated single
copy of HR repair substrate were used. Double
strand break (DSB)-induced HR in these cells
results in restoration and expression of GFP
(35). Cells were transfected with a plasmid
encoding I-SceI endonuclease to induce DSBs.
24 hrs after transfections, cells were exposed to
DMSO or ML364 for 24 hrs. Cells were then
subjected to fluorescence- activated cell sorting
(FACS) analysis to quantify the percentage of
viable GFP-positive cells. The RAD51 foci in
Hela
cells
were
detected
by
immunofluoroscence as described (34) using
anti-RAD51 (EMD Millipore) and Alexa Fluor
488-conjugated secondary antibodies. The
quantification of cells with RAD51 foci was
performed by counting the number of cells with
at least ten RAD51 foci per cell.
Dual-Glo
luciferase
assay-The
ORFeome Luciferase fusion plasmid containing
Cyclin D1 ORFeome in a pCMV-IRES-Renilla
Luciferase-IRES-Gateway-Firefly
Luciferase
(pIRIGF) vector was kindly provided by Dr.
Richard Middleton (Belfer Center for Applied
Cancer Science, Boston, MA, USA). The vector
contains a Cyclin D1 ORFeome fused with
Firefly luciferase (22). Renilla luciferase (RLuc)
encoded by the plasmid is used for
normalization purpose. The expression of Cyclin
D1-Firefly luciferase (FLuc) protein in 293T
cells was confirmed by transfecting the cells
with the plasmid followed by western blotting of
the cell lysates with Cyclin D1 (Santacruz, # SC-
753) and FLuc (Abcam, # ab16466) antibodies.
To investigate the changes in Cyclin D1-Firfely
luciferase protein after treatment with
compounds, the luciferase assay was carried out
in 96-well plate. Briefly, the day before
transfection, HCT116 cells were seeded into 96-
ADME Profile- Stability studies were
conducted by dissolving a 10 mM DMSO stock
of compound into a solution of diubiquitin
(DiUb) IQF assay buffer (2% DMSO, 15%
MeCN, 83% DiUb assay buffer). The amount of
remaining compound was monitored through 15
µL injections using LCMS analysis and
determining the area under curve (AUC) of the
corresponding peak (monitored at wavelength
254 nm) at time points of 0 and 48 hours.
ML364 solubility in aqueous solution and assay
buffer, logD, PAMPA permeability and stability
in rat microsomes and mouse plasma, and mouse
plasma were tested following methods described
previously (36,37). Data in mouse and human
10

USP2 inhibition impacts cyclin D1 and cell cycle
microsomes were obtained from Pharmaron, Inc
(Irvine, CA, USA).
Acknowledgements: We are grateful for the technical support from NanoTemper Technologies and
IntelliCyt Corp. We thank Lesley Mathews for useful discussions on cell fixing protocols and flow
cytometry data collection. We acknowledge Ed Kerns, Amy Wang and Kimloan Nguyen for the ADME
and in vitro PK data and helpful discussions, and Tim Foley, Larissa Sambel, Chunyu Yang, and Debbie
Powell for technical assistance. We acknowledge Ya-qin Zhang for assistance with Acumen data
collection.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
Author contributions: MD, RP, JF, MB, AS, DA, KP, ADD, designed research; MD, RP, JF, CT, LL,
LM, HN, CM, PS, KP, EG performed research; MD, RP, JF, MS, ZL, MB, AS, EG, KP, AD analyzed
data; LD, DC provided crucial reagents; MD, RP, JF, AS, MB, KP, MH wrote the paper. All authors
reviewed the results and approved the final version of the manuscript.
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FOOTNOTES
This research was supported by the NCATS Intramural Research Program, Molecular Libraries Initiative
of the NIH Common Fund, National Institutes of Health (U54 MH 084681), Alabama Drug Discovery
Alliance, NIH/NCI grant 5P30CA13148-37, and Translational Research Grant from the Leukemia &
Lymphoma Society (6237-13).
The content is solely the responsibility of the authors and does not necessarily represent the official views
of the National Institutes of Health.
The abbreviations used are: USP2, ubiquitin-specific protease 2; USP8, ubiquitin-specific protease 8;
IQF, internally quenched fluorescence; Di-Ub, di-ubiquitin; DUB, deubiquitinase; DMF, N,N-
dimethylformamide; qHTS, quantitative high-throughput screening; DMEM, Dulbecco’s Minimal Eagle
Medium; DMSO, dimethyl sulfoxide; AUC, area under curve
FIGURE LEGENDS
13

USP2 inhibition impacts cyclin D1 and cell cycle
FIGURE 1. ML364 binds to USP2 and inhibits its activity, while a related chemical analogue 2 does not.
A, Chemical structures of ML364 and 2. B, Plot of inhibition of USP2 biochemical activity of ML364
and 2, assessed using K48- and K63-linked IQF Di-Ub substrates. Colors indicate the compound/substrate
combinations, as follows: Blue is ML364/K48-4, green is ML364/ K63-3, orange is 2/ K48-4 and red is 2
/K63-3. C, Inhibition of activity of caspase 6 (orange), caspase 7 (purple), MMP1 (red), MMP9 (yellow),
USP8 (green) and USP15 (blue) by ML364. D-F, Demonstration of ML364 binding to USP2 through
microscale thermophoresis. Microscale thermophoresis curves (Normalized fluorescence vs. time (s)) and
resultant concentration responses plotted as log (concentration) in M versus normalized thermophoresis
without temperature jump ((average hot)/(average cold)*1000 where hot is the average value between the
pink vertical lines and cold is the average between the blue vertical lines) for (D) ML364 and (E) 2. LED
was 20% and laser power was 17%. F, Microscale thermophoresis curves (Normalized fluorescence vs.
time (s)) and resultant IC50 curves plotted as log (concentration, M) versus normalized thermophoresis
without temperature jump ((average hot)/(average cold)*1000 where hot is the average value between the
pink vertical lines and cold is the average between the blue vertical lines) for titration of USP2 alone.
LED= 20% and MST power 60%. G, ML364 and 2 bind reversibly to USP2. The fluorescence over time
is plotted for ML364 (blue), 2 (red), enzyme alone (green) and no enzyme (purple).
FIGURE 2. Kinomescan visualization for ML364 (10 µM) tested for binding to 102 kinases.
FIGURE 3. ML364 reduces Cyclin D1 protein levels in a time-, dose-, and proteasome-dependent
manner in HCT116 cells and Mino cells. A, HCT116 cells were treated with 10 µM ML364 for the
indicated amount of time (top panel) or with the indicated concentration of ML364 (bottom panel) for 4
hours. B, HCT116 cells were treated with 10 µM 2 for the indicated amount of time (top panel) or with
the indicated concentration of 2 (bottom panel) for 4 hours. C, HCT116 cells were treated with 10 µM
ML364 or 10 µM 2 for 4 hours in the absence or presence of 10 µM MG132. D, Mino cells were treated
with 10 µM ML364 for the indicated amount of time (top panel) or with the indicated concentration of
ML364 (bottom panel) for 4 hours. E, Mino cells were treated with 10 µM 2 for the indicated amount of
time (top panel) or with the indicated concentration of 2 (bottom panel) for 4 hours. F, Mino cells were
treated with 10 µM ML364 or 10 µM 2 for 4 hours in the absence or presence of 10 µM MG132. Cyclin
D1 and USP2 protein levels were assessed by western blotting using a GAPDH control. Bands were
quantitated by ImageJ.
FIGURE 4. ML364 directly targets Cyclin D1 for degradation. A, The effect of ML364 on Cyclin D1
protein levels is confirmed by in-cell ELISA. HCT116 cells were treated with the indicated concentration
of ML364 or 2 for 24 hours, and Cyclin D1 protein levels were quantified by in-cell ELISA. B-C,
Expression of Cyclin D1-FLuc fusion protein in 293-T cells. Cells were transfected with a plasmid vector
encoding Cyclin D1-Fluc and RLuc. 48 hrs after transfection, Cyclin D1-FLuc fusion protein levels were
detected by western blotting using a tubulin control. D, ML364 downregulates Cyclin D1 in HCT116
cells. Twenty four hrs after transfection with a plasmid vector encoding Cyclin D1-Fluc and RLuc (as
indicated in panels b and c), the cells were exposed to ML364. 24 hrs after ML364 exposure, cellular
FLuc and RLuc activity was measured. The fold change of FLuc/RLuc ratio normalized to corresponding
to DMSO-treated cells is shown. E, HSP90 inhibitor BIIB021 reduces FLuc activity in HCT116 cells.
The cells were treated and analyzed as described in (D). F, ML364 downregulates Cyclin D1 in 293T
cells. Fourty eight hrs after transfection with a plasmid vector encoding Cyclin D1-Fluc and RLuc (as
indicated in panels b and c), the cells were exposed to ML364. Four hrs after ML364 exposure, cellular
FLuc and RLuc activity was measured as described in (D).
14

USP2 inhibition impacts cyclin D1 and cell cycle
FIGURE 5. ML364 induces cell cycle arrest and inhibits cell growth in HCT116 and Mino cells. A,
HCT116 and (B) Mino cells were treated with DMSO (purple), 10 µM ML364 + 100 nM nocodazole
(green), or 10 µM 2 + 100 nM nocodazole (red) or 100 nM nocodazole (blue) for 16 hours. The
percentage of cells in each phase of the cell cycle was determined by flow cytometry and is displayed in
each plot below the population brackets, which are, from left to right, G1, S, and G2/M phases,
respectively. C, The percentage of G1 phase (circles) and G2 phase (triangles) for ML364 as determined
by cell image analysis of fixed and stained HCT116 cells treated with ML364. The concentration-
response curves and images of the 6-well plate for the colony forming assays with HCT116 cells for
ML364 (D) and (E) and 2 (F) and (G), respectively.
FIGURE 6. ML364 exposure decreases cell viability and promotes Cyclin D1 degradation in cancer cell
lines. A, The effect of ML364 on cell viability of Mino (squares) and HCT116 (circles) cells as measured
by ATP content using Cell titer GloTM. B-C, The effect of ML364 on cell viability of LnCAP (circles) and
MCF7 (squares) cells as measured by ATP content using Cell titer GloTM. The cells were exposed to
ML364 for 24 hrs (B) or 48 hrs (C). D, ML364 promotes degradation of Cyclin D1 in LnCAP cells and
MCF7 cells. Cells were treated with ML364 for 24 hrs and Cyclin D1 protein levels were assessed by
western blotting using a tubulin control. E-F, ML364 exposure results in a moderate decrease in FAS
levels. LnCAP cells were treated with ML364 for 24 hrs and FAS, MDM2 and Cyclin D1 protein levels
were assessed by western blotting using a vinculin control.
FIGURE 7. Inhibition of USP2 activity by ML364 causes a decrease in HR-mediated DNA repair. A,
U2OS-DRGFP cells were transfected with a plasmid encoding I-SceI endonuclease and cultured for
24 hrs. followed by exposure to ML364 at the indicated concentration for 24 h. Cells were then
subjected to flow cytometric analysis. The relative GFP positive cells normalized by solvent vehicle
treated group are shown. B, C, Hela cells were treated with ML364 (5 µM) for 24 hrs before exposing
them with IR (10 Gy). Eight hrs after IR, cells were analyzed for RAD51 foci by immunofluroscence.
Representative images (B) and quantification (C) of RAD51 foci are shown. At least one hundred cells
were scored for RAD51 foci for each replicate and three replicates were scored.
FIGURE 8. Schematic showing the impact of ML364 on USP2 which leads to accelerated Cyclin D1
degradation and subsequent G0/G1 cell cycle arrest.
FIGURE 9. A, Synthetic route to ML364. B, Synthetic route to compound 2.
15

USP2 inhibition impacts cyclin D1 and cell cycle
TABLES
Table 1. Summary of Biochemical and Cell Assay Results
ML364
Compound 2a
USP2 Di-Ub IQF K48-4 (IC50)
USP2 Di-Ub IQF K63-3 (IC50)
HCT116 Cell Viability (EC50)
Mino Cell Viability (EC50)
HCT116 Clonogenic (EC50)
1.1 µM
1.7 µM
3 µM
inactive
inactive
inactive
inactive
inactive
inactive
4.7 µM
3.6 µM
HCT116 Acumen Cell Cycle (EC50)
HCT116 Flow, Cell cycle
2 µM
inactive
inactive
inactive
arrest in G1
arrest in G1
Mino Flow, Cell cycle
Western HCT116, Cyclin D1
destabilization
0.97 µM
8.5 µM
inactive
inactive
Western Mino, Cyclin D1 destabilization
Caspase 6 (IC50)
inactive
inactive
0.95 µM
inactive
inactive
inactive
inactive
inactive
Caspase 7 (IC50)
USP8 Di-Ub IQF K48-2 (IC50)
USP20 Di-Ub IQF K48-2 (IC50)
MMP1 (IC50)
ND
MMP9 (IC50)
inactive
inactive
ND
ND
102 Kinases Screened at 10 µM
a ND= Not determined
16

USP2 inhibition impacts cyclin D1 and cell cycle
Table 2. Kinase Competition Binding for ML364 from Kinomescan
Entrez Gene
Symbol
Percent
Control
Entrez Gene
Symbol
Percent
Control
Entrez Gene
Symbol
Percent
Control
ABL1
ABL1
100
82
EPHA2
ERBB2
ERBB4
MAPK3
PTK2
90
100
99
100
100
85
88
71
100
88
83
79
81
75
98
88
84
84
97
85
96
100
73
81
83
89
84
90
69
100
92
92
95
99
PAK2
PAK4
100
98
ABL1
90
CDK16
PDGFRA
PDGFRB
PDPK1
PIK3C2B
PIK3CA
PIK3CG
PIM1
96
ABL1
90
91
ACVR1B
CABC1
AKT1
94
95
84
FGFR2
FGFR3
FLT3
100
79
92
AKT2
56
87
ALK
68
GSK3B
IGF1R
89
AURKA
AURKB
AXL
87
94
87
CHUK
PIM2
100
90
62
IKBKB
INSR
PIM3
BMPR2
BRAF
85
PRKACA
PLK1
95
82
JAK2
98
BRAF
94
JAK3
PLK3
78
BTK
84
MAPK8
MAPK9
MAPK10
KIT
PLK4
83
CAMK2B
CDK19
CDK2
93
PRKCE
RAF1
88
100
100
88
93
RET
92
CDK3
KIT
RIOK2
ROCK2
RPS6KA3
NUAK2
SRC
100
89
CDK4
88
KIT
CDK4
81
STK11
MAP3K4
MAPKAPK2
MARK3
MAP2K1
MAP2K2
MET
100
72
CDK5
100
76
CDK7
96
CDK8
100
93
SRPK3
TGFBR1
TEK
96
CDK9
100
98
CHEK1
CSF1R
CSNK1D
CSNK1G2
DCLK1
DYRK1B
EGFR
84
100
100
98
NTRK1
TSSK1B
TYK2
100
44
MKNK1
MKNK2
MAP3K9
MAPK14
MAPK11
PAK1
83
100
100
86
ULK2
81
KDR
79
EGFR
95
17

USP2 inhibition impacts cyclin D1 and cell cycle
Table 3. ADME Properties of ML364
a
Aq. Kinetic
Solubility
Aq. Kinetic
Solubility (pH 8,
DiUb Assay
Aq. Stability (2%
DMSO, 15%
MeCN, 83% pH 8
Log D
PAMPA
(measured at Permeabilitya
pH 7.4, PBS
(pH 7.4, PBS
Buffer)a
Buffer without β- DiUb assay buffer)
Buffer)b
ME)a
ML364
b
<2 µM
28.9 µM
>48 hrs
2.31
82.2 x 10-6
cm/s
Rat Microsomal
Mouse
Microsomal
Stability,
Human
Microsomal
Stability,
Mouse Plasma
Stability
a
Stability, t
½
(remaining at 30
(remaining at 30
(remaining at 30
minutes)a
minutes)c
minutes)c
ML364
15 minutes
61 %
84 %
100 %
a Performed in-house.; b Contracted to Analiza, Inc.; c Contracted to Pharmaron, Inc.
18

USP2 inhibition impacts cyclin D1 and cell cycle
FIGURES
Fig. 1
19

USP2 inhibition impacts cyclin D1 and cell cycle
Fig. 2
20

USP2 inhibition impacts cyclin D1 and cell cycle
Fig. 3
21

USP2 inhibition impacts cyclin D1 and cell cycle
Fig. 4
22

USP2 inhibition impacts cyclin D1 and cell cycle
Fig. 5
23

USP2 inhibition impacts cyclin D1 and cell cycle
Fig. 6
24

USP2 inhibition impacts cyclin D1 and cell cycle
Fig. 7
25

USP2 inhibition impacts cyclin D1 and cell cycle
Fig. 8
26

USP2 inhibition impacts cyclin D1 and cell cycle
Fig. 9
A
B
27

Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates Cyclin
D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell
Lymphoma Models
Mindy I. Davis, Rajan Pragani, Jennifer T. Fox, Min Shen, Kalindi Parmar, Emily F.
Gaudiano, Li Liu, Cordelle Tanega, Lauren McGee, Matthew David Hall, Crystal
McKnight, Paul Shinn, Henrike Nelson, Debasish Chattopadhyay, Alan D. D'Andrea,
Douglas S. Auld, Larry J. DeLucas, Zhuyin Li, Matthew B. Boxer and Anton Simeonov
J. Biol. Chem. published online September 28, 2016
Access the most updated version of this article at doi: 10.1074/jbc.M116.738567
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