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Supporting Information
Centrosomal Kinase Nek2 Cooperates with Oncogenic Pathways to Promote
Metastasis
Tirtha K. Das4, Dibyendu Dana1, Suneeta S. Paroly1, Senthil K. Perumal2, Satyakam
Singh3, Hugo Jhun1, Jay Pendse4, Ross L. Cagan4, Tanaji T. Talele3, Sanjai Kumar1,*
1
Department of Chemistry and Biochemistry, Queens College and the Graduate Center of
the City University of New York, Queens, New York 11367-1597, USA
2
Department of Chemistry, The Pennsylvania State University, University Park, PA
16802, USA
3
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St.
John's University, Queens, NY 11439, USA
4
Department of Developmental and Regenerative Biology, Mount Sinai School of
Medicine, New York, New York 10029, USA
*Corresponding author: Tel: 718-997-4120, Fax: 718-997-5531
Email: [email protected]
1. Abbreviations
NMP - N-methyl-2-pyrrolidone; TIS – Triisopropylsilane; BOP - Benzotriazol-1yloxy)tris(dimethylamino)phosphonium
Hydroxybenzotriazole;
hexafluorophosphate;
HoBT
-
1-
DTT – Dithiothreitol; DMSO - Dimethyl sulfoxide; BSA -
Bovine serum albumin;
2. (a) The sequence alignments of serine/threonine kinase hNek2, and receptor tyrosine
kinase EGFR and HER2 kinases is shown in Figure S.1 below.
(b) Computational Validation of In Silico Docking Methodology Using Existing
Nek2 Inhibitors:
To ensure that our in silico docking methodology would yield a reasonable outcome, a
two-pronged approach was undertaken. First, we validated the ability of our docking
algorithm to reproduce the co-crystallized pose of the pyrroleindolinone Nek2 inhibitor.
Indeed a good agreement between the docked and the crystal structure conformation was
observed, as evident from the root mean square (rms) deviation values of 0.20 Å when
heavy atoms were superimposed, and 1.14 Å, when heavy atoms along with hydrogen
atoms were superimposed. This confirmed the reliability of the Glide docking procedure
in reproducing the experimentally observed binding mode for Nek2 inhibitor and instilled
confidence that the parameters set for Glide docking were reasonable to provide a
meaningful insight into the predicted binding mode of EGFR and HER2 inhibitor within
the active site of Nek2.
To further validate our computational approach, we undertook a second approach.
We did this to establish if our Glide docking methodology would be able to discriminate
between the reported potent and weakly active Nek2 inhibitors. We therefore carried out
in silico docking experiments on a “validation set” of 10 reported Nek2 inhibitors (For
structure and their reported IC-50 values see Table S1). These included eight potent Nek2
inhibitors exhibiting IC-50 of 20 nM to 570 nM and two moderately potent Nek2
inhibitors with IC-50 of 1300 nM and 4900 nM. A good correlation in the reported
experimental IC-50 values and their corresponding Glide Scores (reported in kCal/mol)
validated the correctness of our docking algorithm. Notably, this investigation ranked
four of the most potent Nek2 inhibitors reported to date as top hits with the Glidescores
ranging from -6.9 to -8.1 kcal/mol (Table S1). This process instilled confidence in our
virtual screening methodologies, and allowed us to move forward with the virtual
screening of existing EGFR/HER2 inhibitors as potential Nek2 inhibitors.
Table S1. Validation of Our Computational Docking Strategy Using 10 known Nek2
inhibitors
Structures and Chemical Names of Known Sets of
Glide-Score
IC-50
Existing Nek2 Kinase Inhibitors
(kcal/mol)
(nM)
S.N.
O
Cl
N
1
HN
-8.07
20
-6.93
25
-7.99
60
-7.53
130
N
O
H
5-(2-Chloro-acetyl)-3-(5-methyl-3H-imidazol4-ylmethylene)-1,3-dihydro-indol-2-one
N
N
O
S
NH2
O
O
2
F
N
F
F
5-[6-(1-Methyl-piperidin-4-yloxy)-benzoimidazol-1-yl]-3[1-(2-trifluoromethyl-phenyl)-ethoxy]-thiophene-2-carboxylic acid amide
O
Cl
N
3
HN
N
O
H
5-(2-Chloro-acetyl)-3-(3H-imidazol-4-ylmethylene)-1,3-dihydroindol-2-one
O
Cl
N
4
HN
N
O
H
5-(2-Chloro-acetyl)-3-(2-ethyl-5-methyl-3H-imidazol
-4-ylmethylene)-1,3-dihydro-indol-2-one
O
5
N
NH2
N
N
OH
O
-7.50
200
-7.70
310
-7.05
380
-6.95
570
O
O
1-[3-Amino-6-(3,4,5-trimethoxy-phenyl)-pyrazin-2-yl]
-2,3-dimethyl-piperidine-4-carboxylic acid
S
N
6
N
H
N
O
N
NH
N
N
O
N-(3-{5-[2-(3-Morpholin-4-yl-phenylamino)-pyrimidin-4-yl]
-imidazo[2,1-b]thiazol-6-yl}-phenyl)-2-phenyl-acetamide
N
N
NH2
O
O
7
O
F
F
F
N
4-[5-(3-Dimethylamino-propoxy)-benzoimidazol-1-yl]
-2-[1-(2-trifluoromethyl-phenyl)-ethoxy]-benzamide
N
N
O
8
NH2
O
O
F F
F
N
4-[5-(1-Methyl-piperidin-4-yloxy)-benzoimidazol-1-yl]
-2-[1-(2-trifluoromethyl-phenyl)-ethoxy]-benzamide
O
O
O
O
HO
N
9
O
-5.85
1300
-5.15
4900
N
H2 N
4-[3-Amino-6-(3,4,5-trimethoxy-phenyl)
-pyrazin-2-yl]-2-methoxy-benzoic acid
O
Cl
N
10
HN
N
O
5-(2-Chloro-acetyl)-3-(2-ethyl-5-methyl-3H-imidazol4-ylmethylene)-1-methyl-1,3-dihydro-indol-2-one
(c) Selection Criteria for Choosing Seven Potential Nek2 Inhibitory Candidates
from Focused EGFR/HER2 Inhibitory Library:
The presence of key interactions in docked structures previously reported for
inhibitor-bound Nek2 structure was the primary criterion for assigning compounds a high
score (1-3). Some of these key interactions included hydrogen bonding interaction with
the backbone of Cys89 and Asp159, electrostatic interaction with Lys37, ionic interaction
with Asp93, hydrophobic interaction with Met86, Phe148, and Tyr88, and packing of
aromatic ring against α-T helix residues. This analysis resulted in selection of 14
compounds from the library for further evaluation. A visual inspection of these 14 virtual
hits with respect to presence of substituents at the 4-, 6- and 7-positions (exception
lapatinib) of the quinazoline or quinoline ring (hinge loop binding scaffold), Michael
acceptor alone and/or capped with basic tertiary amine group, basic amine group
substituent instead of Michael acceptor at 6-position of the quinoline or quinazoline ring
resulted in selection of 7 representative compounds (See Table S2 below) for in-vitro
screening.
Table S2. Structures of the Potential Nek2-Inhibitory Lead Compounds Identified
from the In Silico Screening of Existing EGFR/HER2 Inhibitors
Glide-Score
S.N.
Structures and Chemical Name
(kcal/mol)
O
N
HN
N
O
1
N
HN
Cl
-7.93
O
NERATINIB
N
4-Dimethylamino-but-2-enoic acid {4-[3-chloro-4-(pyridin-2-ylmethoxy)phenylamino]-3-cyano-7-ethoxy-quinolin-6-yl}-amide
O
N
HN
N
2
O
N
HN
Cl
-7.86
PELITINIB
F
4-Dimethylamino-but-2-enoic acid [4-(3-chloro-4-fluorophenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide
N
N
H
N
3
O S
O
HN
Cl
O
O
LAPATINIB
F
[3-Chloro-4-(3-fluoro-benzyloxy)-phenyl]-(6-{5-[(2-methanesulfonyl-ethylamino)-methyl]-furan-2-yl}-quinazolin-4-yl)-amine
-7.65
O
O
N
H
HN
N
4
N
HN
O
Cl
-7.23
AFATINIB
F
4-Dimethylamino-but-2-enoic acid [4-(3-chloro-4-fluorophenylamino)-7-[{(3S){tetrahydro-furan-3-yloxy}-quinazolin-6-yl]amide
O
N
O
N
N
HN
5
O
CANERTINIB
HN
Cl
-7.38
F
N-[4-(3-Chloro-4-fluoro-phenylamino)-7(3-morpholin-4-yl-propoxy)-quinazolin-6-yl]-acrylamide
O
O
6
N
N
N
O
HN
GEFITINIB
Cl
-7.30
F
(3-Chloro-4-fluoro-phenyl)-[7-methoxy-6(3-morpholin-4-yl-propoxy)-quinazolin-4-yl]-amine
7
O
O
O
O
ERLOTINIB
N
N
HN
-7.52
[6,7-Bis-(3-methoxy-propoxy)-quinazolin4-yl]-(3-ethynyl-phenyl)-amine
3. Expression of Active Wild Type Human Nek2 Kinase
Human Nek2 plasmid was a generous gift from Prof. Andrew M. Fry, University of
Leicester. The supplied plasmid contained Nek2 cDNA in a pGEM-3ZF (-) vector with
ampicillin resistant cassette as the selection marker. Following plasmid amplification in
DH5α cells, we prepared and isolated the plasmid.
Human Nek2 plasmid was a generous gift from Prof. Andrew M. Fry, University
of Leicester. The supplied plasmid contained Nek2 cDNA in a pGEM-3ZF (-) vector with
ampicillin resistant cassette as the selection marker. Following plasmid amplification in
DH5α cells, we prepared and isolated the plasmid.
The gene encoding the wild-type Nek-2 kinase was PCR amplified from a pGEM
plasmid
containing
the
Nek2
gene
CAGGACGTCATATGCCTTCCCGGGCTGAGGACTATG-3′
using
5′-
and
5′-
GACATGCCTCGAGGCGCATGCCCAGGATCTGTCTGC-3′ as the forward and
reverse primers respectively. The underlined sequences denote the NdeI and XhoI
restriction sites, respectively, which were used to clone the PCR amplified product into
the pET32a expression vector. The C-terminally (His)6-tagged Nek2 enzyme was
achieved by incorporating the Nek2 gene into pET32a that supplies a hexahistidine tag at
the C-terminus of the protein connected to the open reading frame of Nek2 by a two
residue amino acid linker. The recombinant plasmids were transformed into E. coli XL-1
Blue cells and clones were selected on nutrient agar plates containing kanamycin (30
μg/ml). The identity of the clones was verified by dideoxy sequencing.
The gene encoding the wild-type lambda protein phosphatase (LPP2) was PCR
amplified
from
the
lambda
genomic
GATATACATATGCGCTATTACGAAAAAATTGA-3′
DNA
using
and
5′5′-
CCAGACTCGAGTCATGCGCCTTCTCCCTGTACCTG-3′ as the forward and reverse
primers respectively. The underlined sequences denote the NdeI and XhoI restriction
sites, respectively, which were used to clone the PCR amplified product into the pET22b
expression vector. The recombinant plasmids were transformed into E. coli XL-1 Blue
cells and clones were selected on nutrient agar plates containing ampicillin (50 μg/ml).
The identity of the clones was verified by dideoxy sequencing. The recombinant Nek2
protein was expressed under IPTG induction by a slight modification of an earlier
published protocol (4).
The vectors containing the Nek2 and LPP2 genes were transformed into BL21
(RIPL) codon plus E. coli cells and single colony selected on a plate containing
kanamycin (30 μg/ml) and ampicillin (50 μg/ml) was used to inoculate a 50 ml Luria
Broth (LB) overnight culture. A 10 ml of the
overnight culture was diluted into two 1L
culture
in
two
liter
flasks
containing
kanamycin (30 μg/ml), chloramphenicol (30
μg/ml) and ampicillin (50 μg/ml). The cells
were grown at 37 °C until the absorbance
reached ~1.0 at 600 nm. The cells were then
cooled down to 18 °C and 0.2 mM isopropyl
1-thio-β-D-galactopyranoside
(IPTG)
and
continued to grow overnight. The cells were
harvested by ultracentrifugationa at 5000 rpm at 4 °C. The harvested cells were then
resuspended in 50 ml of lysis buffer containing 20 mM Tris-HCl (pH: 7.5), 300 mM
NaCl, 5 mM imidazole in the presence of an EDTA-free protease inhibitor cocktail tablet
and lysed by sonication. The cell debris was removed by ultracentrifugation at 15000 rpm
at 4 °C for 30 min. The cell-free lysate thus obtained was loaded on to a Ni-NTA column,
equilibrated with lysis buffer. The protein was eluted with 100 ml of a linear gradient of
imidazole (5-150 mM). The protein fractions were separated on a 12% SDS-PAGE gel,
and the fractions containing the protein were pooled together and dialyzed overnight
against 2L of buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5 mM
EDTA, 2 mM dithiothreitol (DTT), 5% glycerol. The protein was then applied to a SP HP
column at rate of 2 ml/min. The protein was then eluted with a linear gradient of 0.15-0.8
M NaCl in buffer containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM DTT, and
5% glycerol. The fractions containing the protein analyzed by 12% SDS-PAGE were
pooled together and concentrated to 27 µM and frozen aliquots were stored at -80 °C.
Protein concentration was calculated using the extinction coefficient of 34840 M-1 cm-1.
Enzymatic activity of purified hNek2 was evaluated by monitoring the
incorporation of [γ32P]ATP into dephosphorylated β-casein (Sigma-Aldrich Inc.), a
known substrate of hNek2. As anticipated, we observed efficient phosphorylation of βcasein by hNek2 protein (Figure S.3).
4. Synthesis of a Nek2 Peptide Substrate for In vitro Kinase Assay
All chemical reagents and resins for the
synthesis were purchased from Advanced
ChemTech
Inc.,
Louisville
Phospholemma-derived
KY,
Nek2
USA.
peptide
substrate, Ac-IRRLSTRRR-CONH2, was synthesized on solid support using standard
solid phase peptide synthesis (SPPS) protocol (5). Rink amide resin (Creosalus Inc.) with
an amino group loading of 0.62 mmol/g was used. Briefly, the following typical
procedure was adopted for coupling of the first amino acid, arginine to the resin matrix.
The resin (200 mg, 0.124 mmol) was swelled in NMP (3 ml) solvent for five minutes. To
this was added a cocktail of Fmoc-Arg(Pbf)-OH (241 mg, 0.372 mmol), BOP (164.5 mg,
0.372 mmol), HoBt (50.2 mg, 0.372 mmol), and 4-methylmorpholine (125 mg, 1.24
mmol) in NMP (5 ml). The resulting mixture was shaken vigorously at room temperature
for 2 hours. The excess reagents were removed by vacuum filtration and the resin was
thoroughly washed with NMP, isopropanol, and dichloromethane successively (3 times
each). A negative Kaiser test indicated that the amino acid coupling to resin matrix was
successful. The Fmoc-group was then removed using 30% piperidine in NMP (30
minutes shaking) and resin was washed again as described before. Coupling of
subsequent amino acids was performed in a similar manner. Acetylation of N-terminal
isoleucine was performed by addition of acetic anhydride (253.1 mg, 2.48 mmol) and 4methylmorpholine (251 mg, 2.48 mmol) to the resin in NMP (30 min shaking). The resin
was thoroughly washed and the final Nek2 peptide substrate was cleaved using a
cleavage cocktail (95% TFA, 2.5% TIS, and 2.5% water, 6 ml, 3hrs). The volatile
components were evaporated to dryness under nitrogen and the peptide was precipitated
out using cold ethyl ether (15 ml). The crude peptide was dissolved in water and the
purification was achieved on an RP-HPLC (Waters Inc) column, using a linear gradient
of water/acetonitrile containing 0.1% TFA (67% yield). The final characterization of
Nek2 peptide substrate was achieved using electro-spray ionization mass spectrometry
(FLEET, Thermo Scientific Inc.). Expected mass calculated for quadruply-charged parent
ion C51H99N25O12: 314.6; Observed: 314.8).
dd_120424184756 #30 RT: 0.09 AV: 1 NL: 7.56E4
T: ITMS + p ESI Full m s [200.00-1300.00]
314.83
100
95
90
85
80
75
70
65
Relative Abundance
60
55
50
45
40
419.25
35
30
343.08
628.17
25
20
15
10
5
0
200
456.92
252.25
300.58
300
367.08
400
495.00
500
684.58
668.00
550.00 599.58
600
703.75 760.67
700
798.50 855.75 894.00
800
900
973.58
1032.83
1000
1131.67
1100
1204.75 1255.08
1200
1300
m /z
5. In vitro Inhibitor Screening and IC-50 Determination
All in-vitro Nek2 kinase assays were performed in triplicate at 25 °C at pH 7.5 in
kinase assay buffer (50 mM Tris HCl buffer containing 2 mM DTT, 10 mM MgCl2,
0.3mg/ml BSA, 0.5 mM Na2EDTA) using wt human Nek2. All inhibitor stock solutions
were made in DMSO at a net concentration of 10 mM and stored at -20 °C when not in
use. Net DMSO concentrations in all assays were maintained at 5%. Initial screening of
small molecule drug library was performed in a 96-well format at an inhibitory
concentration of 10 μM. Total assay volume in each well was restricted to 50 μl. A
phospholemma-derived
peptide,
Ac-Ile-Arg-Arg-Leu-Ser-Thr-Arg-Arg-Arg-CONH2,
synthesized as described earlier, was used as the hNek2 substrate. The following typical
assay procedure was adopted: To a 46 μl kinase assay buffer solution containing 25 μM
cold ATP supplemented with 2 μCi of [γ-32P] ATP (Perkin Elmer Inc., USA) was added
2.5 μl of Inhibitor solution (net inhibitor concentration 5 μM) and 2.5 μl enzyme (net
hNek2 concentration 20 nM). The plate was then incubated for seven minutes. The
enzymatic reaction was initiated by the addition of 2 μl of Nek2 peptide substrate (net
concentration 50 μM) to the assay mixture. After the reaction was allowed to run for
seven minutes, it was quenched by the addition of 100 μl of 6% phosphoric acid. After
five minutes, 45 μl of the quenched reaction mixture was transferred onto a Whatman 350
Unifilter plate (P81 cellulose phosphate paper) and incubated for thirty minutes. Each
well of the plate was then sequentially washed with 0.1% phosphoric acid (3 × 100 μl)
and water (3 × 100 μl) using a vacuum manifold (Millipore Corp.). After the plate was
dried on vacuum for thirty minutes, 50 μl of scintillation fluid (EcoScintA) was added.
The top and bottom of the plate was sealed using TopSeal A (Perkin Elmer Inc.). The
sealed plate was then loaded into TopCount NXT (Perkin Elmer Inc.) for scintillation
counting. The percentage inhibition was assessed upon direct comparison of counts from
wells that did not have any inhibitor present in it (control well). For determination of IC50 values, similar assay protocols were followed except that relative activity was
determined in a dose-dependent manner. The experimental data thus obtained were
plotted using SigmaPlot software and fitted using Enzyme Kinetics module to obtain IC50 values.
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