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Supporting Information
Novel anticancer agents based on targeting the trimer interface of the
PRL phosphatase
Yunpeng Bai1, Zhi-Hong Yu1, Sijiu Liu2, Lujuan Zhang2, Ruo-Yu Zhang1, Li-Fan Zeng2,
Sheng Zhang1, and Zhong-Yin Zhang1,2,*
1
Department of Medicinal Chemistry and Molecular Pharmacology, Department of
Chemistry, Purdue Center for Cancer Research, and Purdue Center for Drug Discovery,
Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA;
2
Department of Biochemistry and Molecular Biology, Indiana University School of
Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA
*To whom correspondence should be addressed: [email protected].
Running title: PRL detrimerizers as novel anticancer agents
In vitro and in vivo cross-linking experiments. To assess the oligomeric state of
PRL1 in vitro, (His)6-tagged recombinant PRL1 proteins were cross-linked with
glutaraldehyde (Sigma, 25% glutaraldehyde solution, grade I). The reactions were
performed in 20 μL solutions containing 5 μg of protein in phosphate-buffered saline
(PBS, pH 7.5). Recombinant PRL1 was treated with 10 μM compound for 30 min, and
then cross-linked by incubating with 0.005% glutaraldehyde at room temperature for 10
min. The reaction was terminated by addition of pH 7.5 Tris-HCl (final concentration 50
mM) and 5 min incubation on ice. The samples were separated on SDS-PAGE and
analyzed by Coomassie Blue staining. For in vivo cross-linking, HEK293 cells with 90%
confluence were treated with 20 μM compound for 24 hours, and then fixed with 1%
formaldehyde (Thermo, 16% formaldehyde solution) for 10 min at room temperature.
Cells were washed twice in ice-cold PBS and lysed on ice for 30 min following by
immunoprecipitation with HA antibody. The reaction mixtures were analyzed by SDSPAGE and detected by immunoblotting with anti-HA antibody (Santa Cruz). The
detrimerization efficacy of the compounds was determined by percentage of
trimer/monomer ratio reduction.
Synthesis of Cmpd-43 and its analogs. To synthesize methyl 3-(5,6dimethylisoindolin-2-yl)benzoate, a solution of 3-(5,6-dimethylisoindolin-2-yl)benzoic
acid (267 mg, 1 mmol), concentrated H2SO4 (0.5 mmol) in methanol (20 mL) were
heated to reflux overnight. The methanol was removed under vacuum. The mixture was
quenched with saturated NaHCO3 (100 mL) and extracted with EtOAc (3 * 50 mL). The
combined organic extract was dried over sodium sulfate and the solvent was removed in
vacuum. The residue was purified by silica gel column chromatography using
hexane/ethyl acetate (8:1) to provide the title compound as a pale solid (60 mg, 21.3%).
To synthesize Analog-1 (3-(5,6-dimethylisoindolin-2-yl)benzohydrazide), a solution of
methyl 3-(5,6-dimethylisoindolin-2-yl)benzoate (1.4 g, 4.98 mmol), hydrazine hydrate (3
mL) in methanol (20 mL) were heated to reflux for 3 h. Most of the methanol was
removed under vacuum. The residue was washed by water (5 mL) and methanol (5 mL)
to afford title compound as white solid (1.1 g, 78.5%). To synthesize Cmpd-43 and
Analog-5 to Analog-7, to a stirred solution of Analog-1 (20 mg, 0.07 mmol) and
appropriate benzaldehyde (0.15 mmol) in methanol (5 mL), glacial acetic acid (0.1 mL)
was added. The mixture was refluxed for 3 h, after which the solution was cool down,
filtrated and the resulting solid was washed by methanol (2 mL) to afford title compounds
as pale solids.
Assessment of PTP inhibition by Cmpd-43. PTP activity was assayed using pNPP
as a substrate at 25C in 50 mM 3,3-dimethylglutarate buffer, pH 7.0, containing 1 mM
EDTA with an ionic strength of 0.15 M adjusted by NaCl. The assays were performed in
96-well plates in a total reaction volume of 200 μL. The reaction was initiated by the
addition of enzyme to a reaction mixture containing pNPP at a concentration equaling the
Km value for each PTP and 20 M Cmpd-43. The reaction was quenched by the addition
of 5 M NaOH. The nonenzymatic hydrolysis of pNPP was corrected by measuring the
control without the addition of enzyme. The amount of product p-nitrophenol was
determined from the absorbance at 405 nm detected by a Spectra MAX340 microplate
spectrophotometer (Molecular Devices) using a molar extinction coefficient of 18,000 M1
cm-1. In addition to PRL1, other PTPs evaluated included the receptor-like PTPs, PTPμ,
PTPε, LAR, PTPσ and PTPγ, cytosolic PTPs, PTP1B, Lyp, SHP1, PTPH1, HePTP, STEP,
and PEZ, the dual specificity phosphatase VHR, VHZ, MKP5, CDC14A, and the low
molecular weight PTP.
PRL1 expression, purification, crystallization and data collection. Recombinant
PRL1 expression and purification was described previously (1). For co-crystallization of
PRL1 with Analog-3, 100 μL 6 mg/mL PRL1 was mixed with 6 μL stock of Analog-3
(10 mM in DMSO), then crystals were grown by vapor diffusion in hanging drops at 4C,
room temperature, 30C and 37 C respectively. Drops containing 1:1 volumes of protein
in stock buffer and reservoir solutions were equilibrated against the reservoir solution A
(1.9 M ammonium sulfate, 100 mM sodium acetate, pH 4.6 ~ 4.9). The crystals were
observed after 2 weeks (for 37 C), or 7 weeks (for 30 C), or half year (for room
temperature); No crystals were observed under 4 C condition in 8 months. The crystals
(grown at 37 C) was transferred into a reservoir solution B (90% saturated lithium
sulfate, 0.5 mM Analog-3, 5% DMSO), soaked for 1 second, then flash-cooled in liquid
nitrogen. X-ray data were collected at 100 K at SBC-CAT beamline 19-BM at the
Advanced Photon Source (Argonne, IL) equipped with a mosaic CCD detector. The
crystals belong to space group C2221 with the following unit cell parameters: a = 46.07 Å,
b = 76.47 Å and c = 86.87 Å. There is one protein molecule in the asymmetric unit. All
data were processed with HKL3000 (2), and the statistics are provided in Table 1.
Structural determination and refinement. The structure of PRL1Analog-3 was
solved by molecular replacement using the program Molrep (3) . The structure of PRL1
(PDB entry code 1ZCK) (4), without the solvent molecules and other small molecules,
was used as a search model. The map revealed the density for the bound Analog-3
(Figure 4). The structure was refined to 1.90 Å resolution with the program CNS1.1 (5),
first using simulated annealing at 2,500 K, and then alternating positional and individual
temperature factor refinement cycles. Electron density maps were inspected and the
model was modified in WinCoot (6). Finally, water molecules were added gradually as
the refinement progressed. They were assigned in the |Fo| - |Fc| difference Fourier maps
with a 3σ cutoff level for inclusion in the model. The statistics of refinements were also
provided
in
Table
1.
Molecular
graphics
were
prepared
using
Pymol
(www.pymol.sourceforge.net).
Histological studies. Tumors and tissues were fixed in 4% paraformaldehyde
(PFA) overnight at 4 °C, embedded in paraffin, serially sectioned (7 μm), and stained
with H&E according to standard methods. For immunohistochemistry, de-paraffined and
hydrated sections were subjected to antigen retrieval by boiling in 10 mM sodium citrate
for 20 min. Sections were then incubated with diluted antibodies (1:50-1:400) at 4 °C
overnight. Signals were detected by VECTASTAIN Elite ABC kit and developed using
DAB substrate from Vector laboratory (Burlingame, CA). Antibodies used were Ki67
(Thermo Fisher Scientific, MA) and cleaved PARP (Cell Signaling Technology, MA).
Images were captured on a Leica DM2500 stereomicroscope. All images are
representative of at least 3 samples.
References
1. Bai Y, Luo Y, Liu S, Zhang L, Shen K, Dong Y, et al. PRL-1 protein promotes
ERK1/2 and RhoA protein activation through a non-canonical interaction with the
Src homology 3 domain of p115 Rho GTPase-activating protein. J Biol Chem
2011;286:42316–24.
2. Minor W, Cymborowski M, Otwinowski Z, Chruszcz M. HKL-3000: the integration of
data reduction and structure solution--from diffraction images to an initial model
in minutes. Acta Crystallogr D Biol Crystallogr 2006;62:859–66.
3. Vagin A, Teplyakov A. MOLREP: an automated program for molecular replacement.
Journal of Applied Crystallography 1997;30:1022–5
4. Sun JP, Wang WQ, Yang H, Liu S, Liang F, Fedorov AA, et al. Structure and
biochemical properties of PRL-1, a phosphatase implicated in cell growth,
differentiation, and tumor invasion. Biochemistry 2005;44:12009–21.
5. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, et al.
Crystallography & NMR system: A new software suite for macromolecular
structure determination. Acta Crystallographica Section D-Biological
Crystallography 1998;54:905–21.
6. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta
Crystallogr D Biol Crystallogr 2010;66:486–501.
Supplemental Figure 1
Supplemental Figure 1. Cmpd-43 selectively inhibits wild-type MEF cells against PRL1-/MEF cells. *p<0.05 (Student's t test), data represent mean (SD) value from 3 independent
experiments.
Supplemental Figure 2
Supplemental Figure 2. Cmpd-43 showed less cell toxicity towards non-tumor cell lines.
A. Compared to breast cancer cell line MCF7, the cell toxicity of Cmpd-43 was
significantly attenuated towards MCF10A cells. B. Compared to MeWo cells, the cell
toxicity of Cmpd-43 was significantly compromised to MEF cells.
Supplemental Figure 3
Supplemental Figure 3. Both PRL2 and PRL3 could also form trimer, and Cmpd-43
could disrupt PRL2 and PRL3 trimerization. A. Amino acid sequence alignment of all
PRLs. Residues involved in trimer interface were labeled as bold letters; while residues
involved in Analog-3 binging were labeled as boxed letters. B. PRL2 and PRL3
trimerization in the presence of either Cmpd-43 or Analog-1 by in vitro crosslinking
assay.
Supplemental Figure 4
Supplemental Figure 4. Knocking down of both PRL1 and PRL2 significantly reduced
both ERK1/2 and Akt activation in MeWo cells.
Supplemental Figure 5
Supplemental Figure 5. Gross melanoma images after 20 days treatment of Cmpd-43.
Supplemental Figure 6
Supplemental Figure 6. In vivo toxicity of Cmpd-43. A. 21 days of Cmpd-43 treatment
(15 mg/kg or 30 mg/kg) showed no significant toxicity on major organs. B. The
histological analysis indicates that 30 mg/kg of Cmpd-43 treatment for 20 days showed
no toxicity.